Chimeric oligomeric compounds comprising alternating regions of northern and southern conformational geometry

ABSTRACT

The present invention relates to novel chimeric oligomeric compounds having a plurality of alternating regions having either RNA like having northern or 3′-endo conformational geometry (3′-endo regions) or DNA like having southern or C2′-endo/O4′-endo conformational geometry. The oligomeric compounds of the present invention have shown reduction in mRNA levels in multiple in vitro and in vivo assay systems and are useful, for example, for investigative and therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/388,856 filed Feb. 19, 2009, which is a continuation of U.S.application Ser. No. 10/936,273, filed Sep. 8, 2004, which claimspriority to U.S. Provisional Application No. 60/501,719 filed Sep. 9,2003 and to U.S. Provisional Application No. 60/568,489 filed May 6,2004, each which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCORE0026USC2SEQ_ST25, created May 30, 2014, which is 564 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel chimeric oligomeric compoundshaving regions of nucleosides that are RNA like having northern or3′-endo conformational geometry (3′-endo regions) and regions ofnucleosides that are DNA like having southern or C2′-endo/04′-endoconformational geometry. In certain embodiments the nucleosides thatcomprise the DNA like regions are 2′-deoxyribonucleosides. Chimericoligomeric compounds include those having 3′-endo regions positioned atthe 3′ and 5′-termini with at least two internal C2′-endo/04′-endoregions that are separated by at least one 3′-endo region. In otherembodiments there are at least 5 separate regions that alternate betweenC2′-endo/04′-endo and 3′-endo regions. The oligomeric compounds of thepresent invention are useful in the regulation of gene expression. Theoligomeric compounds of the present invention have shown reduction inmRNA levels in multiple in vitro and in vivo assay systems. The chimericoligomeric compounds of the present invention are useful, for example,for investigative and therapeutic purposes.

BACKGROUND OF THE INVENTION

Nearly all disease states in multicellular organisms involve the actionof proteins. Classic therapeutic approaches have focused on theinteraction of proteins with other molecules in efforts to moderate theproteins' disease-causing or disease-potentiating activities. In newertherapeutic approaches, modulation of the production of proteins hasbeen sought. A general object of some current therapeutic approaches isto interfere with or otherwise modulate gene expression.

One method for inhibiting the expression of specific genes involves theuse of oligonucleotides, particularly oligonucleotides that arecomplementary to a specific target messenger RNA (mRNA) sequence. Due topromising research results in recent years, oligonucleotides andoligonucleotide analogs are now accepted as therapeutic agents holdinggreat promise for therapeutic and diagnostic methods.

Oligonucleotides and their analogs can be designed to have particularproperties. A number of chemical modifications have been introduced intooligomeric compounds to increase their usefulness as therapeutic agents.Such modifications include those designed to increase binding affinityto a target strand, to increase cell penetration, to stabilize againstnucleases and other enzymes that degrade or interfere with the structureor activity of the oligonucleotide, to provide a mode of disruption(terminating event) once the oligonucleotide is bound to a target, andto improve the pharmacokinetic properties of the oligonucleotide.

Despite these advances, a need exists in the art for the development ofmeans to improve the binding affinity and nuclease resistance propertiesof oligomeric compounds. The present invention meets these needs as wellas other needs.

SUMMARY OF THE INVENTION

The present invention provides chimeric oligomeric compounds comprisingfrom about 5 to about 80 linked nucleosides wherein the chimericoligomeric compounds are divided into at least 5 separate regions andeach of these regions is a continuous sequence of from 1 to about 5nucleosides each having a 3′-endo sugar conformational geometry or acontinuous sequence of from 1 to about 5 2′-deoxyribonucleosides andwherein each of these regions comprising from 1 to about 52′-deoxyribonucleosides is internally located between two of saidregions comprising 1 to about 5 nucleosides each having a 3′-endo sugarconformational geometry or at one of the 3′ or 5′ termini.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows scheme I depicting iterative synthesis of compound 10.

FIG. 2 shows scheme I (continued) depicting iterative synthesis ofcompound 17.

FIG. 3 shows scheme I (continued) depicting iterative synthesis ofcompound I.

FIG. 4 shows scheme II depicting an alternate iterative synthesis ofcompound I starting with compound 20.

FIG. 5 shows scheme III depicting iterative synthesis of compound 33.

FIG. 6 shows scheme IV depicting iterative synthesis of compound 40.

FIG. 7 shows scheme V depicting iterative synthesis of compound 47.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel chimeric oligomeric compoundscomprising regions that alternate between 3′-endo sugar conformationalgeometry (3′-endo regions) and 2′-endo/O4′-endo sugar conformationalgeometry (2′-endo regions). Each of the alternating regions comprisefrom 1 to about 5 nucleosides. The chimeric oligomeric compounds canstart (5′-end) or end (3′-end) with either of the 2 regions and can havefrom about 5 to about 20 separate regions. One or more of thenucleosides of the chimeric oligomeric compound can further comprise aconjugate group. In one aspect of the present invention chimericoligomeric compounds have the formula: T₁-(3′-endo region)-[(2′-endoregion)-(3′-endo region)]_(n)-T₂ wherein n is at least two and each T₁and T₂ is independently an optional conjugate group.

Each of the regions can range from 1 to about 5 nucleosides in lengthallowing for a plurality of motifs for oligonucleotides having the samelength. Such as for example a chimeric oligomeric compound of thepresent invention having a length of 20 base pairs (bp) would includesuch motifs as 3-3-2-4-2-3-3,3-4-1-4-1-4-3 and 4-3-1-4-1-3-4 where eachmotif has the same number and orientation of regions (bold anditalicized numbers are 3′-endo regions, unbold and not underlinednumbers are 2′-endo regions and the number corresponding to each regionrepresenting the number of base pairs for that particular region).

A plurality of motifs for the chimeric oligomeric compounds of thepresent invention has been prepared and has shown activity in aplurality of assays against various targets. In addition to in vitroassays some positive data have also been obtained through in vivoassays. A list of motifs is shown below. This list is meant to berepresentative and not limiting.

Motifs # bp's Regions Motif 20 mer 5 1-8-2-8-1 20 mer 5 2-6-4-6-2 20 mer5 2-7-2-7-2 20 mer 5 3-5-4-5-3 20 mer 5 3-6-1-7-3 20 mer 5 3-7-1-6-3 20mer 7 3-3-2-4-2-3-3 20 mer 7 3-4-1-4-1-4-3 20 mer 7 4-3-1-4-1-3-4 18 mer9 2-2-1-3-1-2-1-3-3 20 mer 9 3-2-1-3-1-3-1-3-3 20 mer 93-2-1-3-1-2-1-3-4 18 mer 9 3-3-1-2-1-3-1-2-2 20 mer 9 3-3-1-2-1-3-1-3-320 mer 9 3-3-1-2-1-2-1-3-4 20 mer 9 3-3-1-3-1-2-1-2-4 20 mer 93-3-1-3-1-2-1-3-3 20 mer 9 5-2-1-2-1-2-1-1-5 20 mer 113-2-2-1-2-1-2-1-1-2-3 20 mer 11 3-1-3-1-2-1-2-1-2-1-3 20 mer 113-1-2-1-2-1-2-1-2-1-4 20 mer 11 3-2-1-2-1-2-1-2-1-2-3 20 mer 113-2-1-2-1-3-1-2-1-1-3 20 mer 15 2-1-1-2-1-1-1-1-1-1-1-1-1-3-2 20 mer 153-1-1-1-1-1-1-1-1-1-1-1-1-1-4 20 mer 191-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-2 # = number of 3′-endo nucleosidesin the region (bolded) # = number of 2′-deoxy ribonucleotides in theregion

Compounds of the Invention

The present invention provides chimeric oligomeric compounds that haveat least 5 regions that alternate between 3′-endo and 2′-endo inconformational geometry. The nucleoside or nucleosides of a particularregion can be modified in a variety of ways to give the region either a3′-endo or a 2′-endo conformational geometry. The conformationalgeometry of a selected nucleoside can be modulated in one aspect bymodifying the sugar the base or both the sugar and the base.Modifications include attachment of substituent groups or conjugategroups or by directly modifying the base or the sugar.

The sugar conformational geometry (puckering) plays a central role indetermining the duplex conformational geometry between anoligonucleotide and its nucleic acid target. By controlling the sugarpuckering independently at each position of an oligonucleotide theduplex geometry can be modulated to help maximize desired properties ofthe resulting chimeric oligomeric compound. Modulation of sugar geometryhas been shown to enhance properties such as for example increasedlipohpilicity, binding affinity to target nucleic acid (e.g. mRNA),chemical stability and nuclease resistance.

The present invention discloses novel chimeric oligomeric compoundscomprised of a plurality of alternating 3′-endo and 2′-endo (including2′-deoxy) regions wherein each of the regions are independently fromabout 1 to about 5 nucleosides in length. The chimeric oligomericcompounds can start and end with either 3′-endo or 2′-endo regions andhave from about 5 to about 19 regions in total. The nucleosides of eachregion can be selected to be uniform such as for example uniform2′-O-MOE nucleosides for one or more of the 3′-endo regions and2′-deoxynucleosides for the 2′-endo regions. Alternatively thenucleosides can be mixed such that any nucleoside having 3′-endoconformational geometry can be used in any position of any 3′-endoregion and any nucleoside having 2′-endo conformational geometry can beused in any position of any 2′-endo region. In some embodiments a5′-conjugate group is used as a 5′-cap as a method of increasing the5′-exonuclease resistance but conjugate groups can be used at anyposition within the chimeric oligomeric compounds of the invention.

3′-Endo Regions

The present invention provides chimeric oligomeric compounds havingalternating regions wherein one of the alternating regions has 3′-endoconformational geometry. These 3′-endo regions include nucleosidessynthetically modified to induce a 3′-endo sugar conformation. Anucleoside can incorporate synthetic modifications of the heterocyclicbase, the sugar moiety or both to induce a desired 3′-endo sugarconformation. These modified nucleosides are used to mimic RNA likenucleosides so that particular properties of an oligomeric compound canbe enhanced while maintaining the desirable 3′-endo conformationalgeometry. Properties that are enhanced by using more stable 3′-endonucleosides include but aren't limited to modulation of pharmacokineticproperties through modification of protein binding, protein off-rate,absorption and clearance; modulation of nuclease stability as well aschemical stability; modulation of the binding affinity and specificityof the oligomer (affinity and specificity for enzymes as well as forcomplementary sequences); and increasing efficacy of RNA cleavage. Thepresent invention provides regions of nucleosides modified in such a wayas to favor a C3′-endo type conformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713.Harry-O′ kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tanget al., J. Org. Chem. (1999), 64, 747-754.)

Alternatively, preference for the 3′-endo conformation can be achievedby deletion of the 2′-OH as exemplified by 2′ deoxy-2′F-nucleosides(Kawasaki et al., J. Med. Chem. (1993), 36, 831-841), which adopts the3′-endo conformation positioning the electronegative fluorine atom inthe axial position. Other modifications of the ribose ring, for examplesubstitution at the 4′-position to give 4′-F modified nucleosides(Guillerm et al., Bioorganic and Medicinal Chemistry Letters (1995), 5,1455-1460 and Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or forexample modification to yield methanocarba nucleoside analogs (Jacobsonet al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al.,Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337) alsoinduce preference for the 3′-endo conformation. Along similar lines,3′-endo regions can include one or more nucleosides modified in such away that conformation is locked into a C3′-endo type conformation, i.e.Locked Nucleic Acid (LNA, Singh et al, Chem. Commun. (1998), 4,455-456), and ethylene bridged Nucleic Acids (ENA, Morita et al,Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)

Examples of modified nucleosides amenable to the present invention areshown below in Table 1. These examples are meant to be representativeand not exhaustive.

TABLE 1

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligoncleotides of the presentinvention. The synthesis of numerous of the modified nucleosidesamenable to the present invention are known in the art (see for example,Chemistry of Nucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend,1988, Plenum press., and the examples section below). Nucleosides knownto be inhibitors/substrates for RNA dependent RNA polymerases (forexample HCV NS5B).

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (T_(m)s)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and 04′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a 04′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as, but notlimited to, antisense and RNA interference as these mechanisms requirethe binding of an oligonucleotide strand to an RNA target strand. In thecase of antisense, effective inhibition of the mRNA requires that theantisense DNA have a minimum binding affinity with the mRNA. Otherwise,the desired interaction between the oligonucleotide strand and targetmRNA strand will occur infrequently, resulting in decreased efficacy.

One routinely used method of modifying the sugar puckering is thesubstitution on the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-β-methoxyethyl substituentalso have been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligonucleotideshaving the 2′-MOE modification displayed improved RNA affinity andhigher nuclease resistance. Chimeric oligomeric compounds having 2′-MOEsubstituents in the wing nucleosides and an internal region ofdeoxy-phosphorothioate nucleotides (also termed a gapped oligonucleotideor gapmer) have shown effective reduction in the growth of tumors inanimal models at low doses. 2′-MOE substituted oligonucleotides havealso shown outstanding promise as antisense agents in several diseasestates. One such MOE substituted oligonucleotide is presently beinginvestigated in clinical trials for the treatment of CMV retinitis.

To better understand the higher RNA affinity of 2′-O-methoxyethylsubstituted RNA and to examine the conformational properties of the2′-O-methoxyethyl substituent, two dodecamer oligonucleotides weresynthesized having SEQ ID NO: 1 (CGC GAA UUC GCG) and SEQ ID NO: 2 (GCGCUU AAG CGC). These self-complementary strands have every 2′-positionmodified with a 2′-O-methoxyethyl. The duplex was crystallized at aresolution of 1.7 Angstrom and the crystal structure was determined. Theconditions used for the crystallization were 2 mM oligonucleotide, 50 mMNa Hepes pH 6.2-7.5, 10.50 mM MgCl₂, 15% PEG 400. The crystal datashowed: space group C2, cell constants a=41.2 Å, b=34.4 Å, c=46.6 Å,□=92.4°. The resolution was 1.7 Å at −170° C. The current R=factor was20% (R_(free) 26%).

This crystal structure is believed to be the first crystal structure ofa fully modified RNA oligonucleotide analogue. The duplex adopts anoverall A-form conformation and all modified sugars display C3′-endopucker. In most of the 2′-O-substituents, the torsion angle around theA′-B′ bond, as depicted in Structure II below, of the ethylene glycollinker has a gauche conformation. For 2′-O-MOE, A′ and B′ of StructureII below are methylene moieties of the ethyl portion of the MOE and R′is the methoxy portion.

In the crystal, the 2′-O-MOE RNA duplex adopts a general orientationsuch that the crystallographic 2-fold rotation axis does not coincidewith the molecular 2-fold rotation axis. The duplex adopts the expectedA-type geometry and all of the 24 2′-O-MOE substituents were visible inthe electron density maps at full resolution. The electron density mapsas well as the temperature factors of substituent atoms indicateflexibility of the 2′-O-MOE substituent in some cases.

Most of the 2′-O-MOE substituents display a gauche conformation aroundthe C—C bond of the ethyl linker. However, in two cases, a transconformation around the C—C bond is observed. The lattice interactionsin the crystal include packing of duplexes against each other via theirminor grooves. Therefore, for some residues, the conformation of the2′-O-substituent is affected by contacts to an adjacent duplex. Ingeneral, variations in the conformation of the substituents (e.g. g′ org⁻ around the C—C bonds) create a range of interactions betweensubstituents, both inter-strand, across the minor groove, andintra-strand. At one location, atoms of substituents from two residuesare in van der Waals contact across the minor groove. Similarly, a closecontact occurs between atoms of substituents from two adjacentintra-strand residues.

Previously determined crystal structures of A-DNA duplexes were forthose that incorporated isolated 2′-O-methyl T residues. In the crystalstructure noted above for the 2′-O-MOE substituents, a conservedhydration pattern has been observed for the 2′-O-MOE residues. A singlewater molecule is seen located between O2′, O3′ and the methoxy oxygenatom of the substituent, forming contacts to all three of between 2.9and 3.4 Å. In addition, oxygen atoms of substituents are involved inseveral other hydrogen bonding contacts. For example, the methoxy oxygenatom of a particular 2′-O-substituent forms a hydrogen bond to N3 of anadenosine from the opposite strand via a bridging water molecule.

In several cases a water molecule is trapped between the oxygen atomsO2′, O3′ and OC′ of modified nucleosides. 2′-O-MOE substituents withtrans conformation around the C—C bond of the ethylene glycol linker areassociated with close contacts between OC′ and N2 of a guanosine fromthe opposite strand, and, water-mediated, between OC′ and N3(G). Whencombined with the available thermodynamic data for duplexes containing2′-O-MOE modified strands, this crystal structure allows for furtherdetailed structure-stability analysis of other modifications.

In extending the crystallographic structure studies, molecular modelingexperiments were performed to study further enhanced binding affinity ofoligonucleotides having 2′-O-modifications of the invention. Thecomputer simulations were conducted on compounds of SEQ ID NO: 1, above,having 2′-O-modifications of the invention located at each of thenucleoside of the oligonucleotide. The simulations were performed withthe oligonucleotide in aqueous solution using the AMBER force fieldmethod (Cornell et al., J. Am. Chem. Soc., 1995, 117,5179-5197)(modeling software package from UCSF, San Francisco, Calif.).The calculations were performed on an Indigo2 SGI machine (SiliconGraphics, Mountain View, Calif.).

Further 2′-O-modifications that will have a 3′-endo sugar influenceinclude those having a ring structure that incorporates a two atomportion corresponding to the A′ and B′ atoms of Structure II. The ringstructure is attached at the 2′ position of a sugar moiety of one ormore nucleosides that are incorporated into an oligonucleotide. The2′-oxygen of the nucleoside links to a carbon atom corresponding to theA′ atom of Structure II. These ring structures can be aliphatic,unsaturated aliphatic, aromatic or heterocyclic. A further atom of thering (corresponding to the B′ atom of Structure II), bears a furtheroxygen atom, or a sulfur or nitrogen atom. This oxygen, sulfur ornitrogen atom is bonded to one or more hydrogen atoms, alkyl moieties,or haloalkyl moieties, or is part of a further chemical moiety such as aureido, carbamate, amide or amidine moiety. The remainder of the ringstructure restricts rotation about the bond joining these two ringatoms. This assists in positioning the “further oxygen, sulfur ornitrogen atom” (part of the R position as described above) such that thefurther atom can be located in close proximity to the 3′-oxygen atom(O3′) of the nucleoside.

Another 2′-sugar substituent group that gives a 3′-endo sugarconformational geometry is the 2′-OMe group. 2′-Substitution ofguanosine, cytidine, and uridine dinucleoside phosphates with the 2′-OMegroup showed enhanced stacking effects with respect to the correspondingnative (2′-OH) species leading to the conclusion that the sugar isadopting a C3′-endo conformation. In this case, it is believed that thehydrophobic attractive forces of the methyl group tend to overcome thedestabilizing effects of its steric bulk.

The ability of oligonucleotides to bind to their complementary targetstrands is compared by determining the melting temperature (T_(m)) ofthe hybridization complex of the oligonucleotide and its complementarystrand. The melting temperature (T_(m)), a characteristic physicalproperty of double helices, denotes the temperature (in degreescentigrade) at which 50% helical (hybridized) versus coil (unhybridized)forms are present. T_(m) is measured by using the UV spectrum todetermine the formation and breakdown (melting) of the hybridizationcomplex. Base stacking, which occurs during hybridization, isaccompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands.

Freier and Altmann, Nucleic Acids Research, (1997) 25:4429-4443, havepreviously published a study on the influence of structuralmodifications of oligonucleotides on the stability of their duplexeswith target RNA. In this study, the authors reviewed a series ofoligonucleotides containing more than 200 different modifications thathad been synthesized and assessed for their hybridization affinity andT_(m). Sugar modifications studied included substitutions on the2′-position of the sugar, 3′-substitution, replacement of the 4′-oxygen,the use of bicyclic sugars, and four member ring replacements. Severalnucleobase modifications were also studied including substitutions atthe 5, or 6 position of thymine, modifications of pyrimidine heterocycleand modifications of the purine heterocycle. Modified internucleosidelinkages were also studied including neutral, phosphorus andnon-phosphorus containing internucleoside linkages.

Increasing the percentage of C3′-endo sugars in a modifiedoligonucleotide targeted to an RNA target strand should preorganize thisstrand for binding to RNA. Of the several sugar modifications that havebeen reported and studied in the literature, the incorporation ofelectronegative substituents such as 2′-fluoro or 2′-alkoxy shift thesugar conformation towards the 3′ endo (northern) pucker conformation.This preorganizes an oligonucleotide that incorporates suchmodifications to have an A-form conformational geometry. This A-formconformation results in increased binding affinity of theoligonucleotide to a target RNA strand.

Molecular modeling experiments were performed to study further enhancedbinding affinity of oligonucleotides having 2′-O-modifications. Computersimulations were conducted on compounds having SEQ ID NO: 1, r(CGC GAAUUC GCG), having 2′-O-modifications of the invention located at each ofthe nucleoside of the oligonucleotide. The simulations were performedwith the oligonucleotide in aqueous solution using the AMBER force fieldmethod (Cornell et al., J. Am. Chem. Soc., 1995, 117,5179-5197)(modeling software package from UCSF, San Francisco, Calif.).The calculations were performed on an Indigo2 SGI machine (SiliconGraphics, Mountain View, Calif.).

In addition, for 2′-substituents containing an ethylene glycol motif, agauche interaction between the oxygen atoms around the O—C—C—O torsionof the side chain may have a stabilizing effect on the duplex (Freierand Altmann, Nucleic Acids Research, 1997, 25, 4429-4443). Such gaucheinteractions have been observed experimentally for a number of years(Wolfe et al., Acc. Chem. Res., 1972, 5, 102; Abe et al., J. Am. Chem.Soc., 1976, 98, 468). This gauche effect may result in a configurationof the side chain that is favorable for duplex formation. The exactnature of this stabilizing configuration has not yet been explained.While not wishing to be bound by theory, it may be that holding theO—C—C—O torsion in a single gauche configuration, rather than a morerandom distribution seen in an alkyl side chain, provides an entropicadvantage for duplex formation.

Representative 2′-substituent groups amenable to the present inventionthat give A-form conformational properties (3′-endo) to the resultantduplexes include 2′-O-alkyl, 2′-O-substituted alkyl and 2′-fluorosubstituent groups. Substituent groups can be various alkyl and arylethers and thioethers, amines and monoalkyl and dialkyl substitutedamines. It is further intended that multiple modifications can be madeto one or more nucleosides and or internucleoside linkages within anoligonucleotide of the invention to enhance activity of theoligonucleotide. Tables 2 through 8 list nucleoside and internucleotidelinkage modifications/replacements that have been shown to give apositive εT_(m) per modification when the modification/replacement wasmade to a DNA strand that was hybridized to an RNA complement.

TABLE 2 Modified DNA strand having 2′-substituent groups that gave anoverall increase in T_(m) against an RNA complement: Positive εT_(m)/mod 2′-substituents 2′-OH 2′-O—C₁-C₄ alkyl 2′-O—(CH₂)₂CH₃2′-O—CH₂CH═CH₂ 2′-F 2′-O—(CH₂)₂—O—CH₃ 2′-[O—(CH₂)₂]₂—O—CH₃2′-[O—(CH₂)₂]₃—O—CH₃ 2′-[O—(CH₂)₂]₄—O—CH₃ 2′-[O—(CH₂)₂]₃—O—(CH₂)₈CH₃2′-O—(CH₂)₂CF₃ 2′-O—(CH₂)₂OH 2′-O—(CH₂)₂F 2′-O—CH₂CH(CH₃)F2′-O—CH₂CH(CH₂OH)OH 2′-O—CH₂CH(CH₂OCH₃)OCH₃ 2′-O—CH₂CH(CH₃)OCH₃2′-O—CH₂—C₁₄H₇O₂(—C₁₄H₇O₂ = Anthraquinone) 2′-O—(CH₂)₃—NH₂*2′-O—(CH₂)₄—NH₂* *These modifications can increase the T_(m) ofoligonucleotides but can also decrease the T_(m) depending onpositioning and number (motif dependant).

TABLE 3 Modified DNA strand having modified sugar ring (see structure)that give an overall increase in T_(m) against an RNA complement:

Positive εT_(m)/mod Q —S— —CH₂— Note: In general ring oxygensubstitution with sulfur or methylene had only a minor effect on T_(m)for the specific motiffs studied. Substitution at the 2′-position withgroups shown to stabilize the duplex were destabilizing when CH₂replaced the ring O. This is thought to be due to the necessary gaucheinteraction between the ring O with particular 2′- substituents (forexample —O—CH₃ and —(O—CH₂CH₂)₃—O—CH₃.

TABLE 4 Modified DNA strand having modified sugar ring that give anoverall increase in T_(m) against an RNA complement:

Positive εT_(m)/mod —C(H)R₁ effects OH (R₂, R₃ both = H) CH₃* CH₂OH*OCH₃* *These modifications can increase the T_(m) of oligonucleotidesbut can also decrease the T_(m) depending on positioning and number(motif dependant).

TABLE 5 Modified DNA strand having bicyclic substitute sugarmodifications that give an overall increase in T_(m) against an RNAcomplement: Formula Positive εT_(m)/mod I + II +

TABLE 6 Modified DNA strand having modified heterocyclic base moietiesthat give an overall increase in T_(m) against an RNA complement:Modification/Formula Positive εT_(m)/mod Heterocyclic base 2-thioTmodifications 2′-O-methylpseudoU 7-halo-7-deaza purines7-propyne-7-deaza purines 2-aminoA(2,6-diaminopurine)

(R₂, R₃ = H), R1 = Br C/C—CH₃ (CH₂)₃NH₂ CH₃ Motiffs-disubstitution R₁ =C/C—CH₃, R₂ = H, R₃ = F R₁ = C/C—CH₃, R₂ = H R₃ = O—(CH₂)₂—O—CH₃ R₁ =O—CH₃, R₂ = H, R₃ = O—(CH₂)₂—O—CH₃* *This modification can increase theT_(m) of oligonucleotides but can also decrease the T_(m) depending onpositioning and number (motif dependant).

Substitution at R₁ can be stabilizing, substitution at R₂ is generallygreatly destabilizing (unable to form anti conformation), motiffs withstabilizing 5 and 2′-substituent groups are generally additive e.g.increase stability.

Substitution of the O4 and O2 positions of 2′-O-methyl uridine wasgreatly duplex destabilizing as these modifications remove hydrogenbinding sites that would be an expected result. 6-Aza T also showedextreme destabilization as this substitution reduces the pK_(a) andshifts the nucleoside toward the enol tautomer resulting in reducedhydrogen bonding.

TABLE 7 DNA strand having at least one modified phosphorus containinginternucleoside linkage and the effect on the T_(m) against an RNAcomplement: εT_(m)/mod + εT_(m)/mod − phosphorothioate¹ phosphoramidate¹methyl phosphonates¹ (¹one of the non-bridging oxygen atoms replacedwith S, N(H)R or —CH₃) phosphoramidate (the 3′-bridging atom replacedwith an N(H)R group, stabilization effect enhanced when also have 2′-F)

TABLE 8 DNA strand having at least one non-phosphorus containinginternucleoside linkage and the effect on the T_(m) against an RNAcomplement: Positive ε T_(m)/mod —CH₂C(═O)NHCH₂—* —CH₂C(═O)N(CH₃)CH₂—*—CH₂C(═O)N(CH₂CH₂CH₃)CH₂—* —CH₂C(═O)N(H)CH₂— (motif with 5′-propyne onT's) —CH₂N(H)C(═O)CH₂—* —CH₂N(CH₃)OCH₂—* —CH₂N(CH₃)N(CH₃)CH₂—* * Thismodification can increase the T_(m) of oligonucleotides but can alsodecrease the T_(m) depending on positioning and number (motifdependant). Notes: In general carbon chain internucleotide linkages weredestabilizing to duplex formation. This destabilization was not assevere when double and tripple bonds were utilized. The use of glycoland flexible ether linkages were also destabilizing.

Suitable ring structures of the invention for inclusion as a 2′-Omodification include cyclohexyl, cyclopentyl and phenyl rings as well asheterocyclic rings having spacial footprints similar to cyclohexyl,cyclopentyl and phenyl rings. Particularly suitable 2′-O-substituentgroups of the invention are listed below including an abbreviation foreach:

2′-O-(trans 2-methoxy cyclohexyl)-2′-O-(TMCHL)

2′-O-(trans 2-methoxy cyclopentyl)-2′-O-(TMCPL)

2′-O-(trans 2-ureido cyclohexyl)-2′-O-(TUCHL)

2′-O-(trans 2-methoxyphenyl)-2′-O-(2 MP)

Structural details for duplexes incorporating such 2-O-substituents wereanalyzed using the described AMBER force field program on the Indigo2SGI machine. The simulated structure maintained a stable A-form geometrythroughout the duration of the simulation. The presence of the 2′substitutions locked the sugars in the C3′-endo conformation.

The simulation for the TMCHL modification revealed that the 2′-O-(TMCHL)side chains have a direct interaction with water molecules solvating theduplex. The oxygen atoms in the 2′-O-(TMCHL) side chain are capable offorming a water-mediated interaction with the 3′ oxygen of the phosphatebackbone. The presence of the two oxygen atoms in the 2′-O-(TMCHL) sidechain gives rise to favorable gauche interactions. The barrier forrotation around the O—C—C—O torsion is made even larger by this novelmodification. The preferential preorganization in an A-type geometryincreases the binding affinity of the 2′-O-(TMCHL) to the target RNA.The locked side chain conformation in the 2′-O-(TMCHL) group created amore favorable pocket for binding water molecules. The presence of thesewater molecules played a key role in holding the side chains in thepreferable gauche conformation. While not wishing to be bound by theory,the bulk of the substituent, the diequatorial orientation of thesubstituents in the cyclohexane ring, the water of hydration and thepotential for trapping of metal ions in the conformation generated willadditionally contribute to improved binding affinity and nucleaseresistance of oligonucleotides incorporating nucleosides having this2′-O-modification.

As described for the TMCHL modification above, identical computersimulations of the 2′-O-(TMCPL), the 2′-O-(2 MP) and 2′-O-(TUCHL)modified oligonucleotides in aqueous solution also illustrate thatstable A-form geometry will be maintained throughout the duration of thesimulation. The presence of the 2′ substitution will lock the sugars inthe C3′-endo conformation and the side chains will have directinteraction with water molecules solvating the duplex. The oxygen atomsin the respective side chains are capable of forming a water-mediatedinteraction with the 3′ oxygen of the phosphate backbone. The presenceof the two oxygen atoms in the respective side chains give rise to thefavorable gauche interactions. The barrier for rotation around therespective O—C—C—O torsions will be made even larger by respectivemodification. The preferential preorganization in A-type geometry willincrease the binding affinity of the respective 2′-β-modifiedoligonucleotides to the target RNA. The locked side chain conformationin the respective modifications will create a more favorable pocket forbinding water molecules. The presence of these water molecules plays akey role in holding the side chains in the preferable gaucheconformation. The bulk of the substituent, the diequatorial orientationof the substituents in their respective rings, the water of hydrationand the potential trapping of metal ions in the conformation generatedwill all contribute to improved binding affinity and nuclease resistanceof oligonucleotides incorporating nucleosides having these respective2′-O-modification.

Ribose conformations in C2′-modified nucleosides containing S-methylgroups were examined. To understand the influence of 2′-O-methyl and2′-S-methyl groups on the conformation of nucleosides, we evaluated therelative energies of the 2′-O— and 2′-S-methylguanosine, along withnormal deoxyguanosine and riboguanosine, starting from both C2′-endo andC3′-endo conformations using ab initio quantum mechanical calculations.All the structures were fully optimized at HF/6-31G* level and singlepoint energies with electron-correlation were obtained at theMP2/6-31G*//HF/6-31G* level. As shown in Table 9, the C2′-endoconformation of deoxyguanosine is estimated to be 0.6 kcal/mol morestable than the C3′-endo conformation in the gas-phase. Theconformational preference of the C2′-endo over the C3′-endo conformationappears to be less dependent upon electron correlation as revealed bythe MP2/6-31G*//HF/6-31G* values which also predict the same differencein energy. The opposite trend is noted for riboguanosine. At theHF/6-31G* and MP2/6-31G*//HF/6-31G* levels, the C3′-endo form ofriboguanosine is shown to be about 0.65 and 1.41 kcal/mol more stablethan the C2′ endo form, respectively.

TABLE 9 Relative energies* of the C3′-endo and C2′-endo conformations ofrepresentative nucleosides Continuum HF/6-31G MP2/6-31-G Model Amber dG0.60 0.56 0.88 0.65 rG −0.65 −1.41 −0.28 −2.09 2′-O—MeG −0.89 −1.79−0.36 −0.86 2′-S—MeG 2.55 1.41 3.16 2.43 *energies are in kcal/molrelative to the C2′-endo conformation

Table 9 also includes the relative energies of 2′-O-methylguanosine and2′-S-methylguanosine in C2′-endo and C3′-endo conformation. This dataindicates the electronic nature of C2′-substitution has a significantimpact on the relative stability of these conformations. Substitution ofthe 2′-O-methyl group increases the preference for the C3′-endoconformation (when compared to riboguanosine) by about 0.4 kcal/mol atboth the HF/6-31G* and MP2/6-31G*//HF/6-31G* levels. In contrast, the2′-S-methyl group reverses the trend. The C2′-endo conformation isfavored by about 2.6 kcal/mol at the HF/6-31G* level, while the samedifference is reduced to 1.41 kcal/mol at the MP2/6-31G*//HF/6-31G*level. For comparison, and also to evaluate the accuracy of themolecular mechanical force-field parameters used for the 2′-O-methyl and2′-S-methyl substituted nucleosides, we have calculated the gas phaseenergies of the nucleosides. The results reported in Table 9 indicatethat the calculated relative energies of these nucleosides comparequalitatively well with the ab initio calculations.

Additional calculations were also performed to gauge the effect ofsolvation on the relative stability of nucleoside conformations. Theestimated solvation effect using HF/6-31G* geometries confirms that therelative energetic preference of the four nucleosides in the gas-phaseis maintained in the aqueous phase as well (Table 9). Solvation effectswere also examined using molecular dynamics simulations of thenucleosides in explicit water. From these trajectories, one can observethe predominance of C2′-endo conformation for deoxyriboguanosine and2′-S-methylriboguanosine while riboguanosine and2′-O-methylriboguanosine prefer the C3′-endo conformation. These resultsare in much accord with the available NMR results on2′-S-methylribonucleosides. NMR studies of sugar puckering equilibriumusing vicinal spin-coupling constants have indicated that theconformation of the sugar ring in 2′-S-methylpyrimidine nucleosides showan average of >75% S-character, whereas the corresponding purine analogsexhibit an average of >90% S-pucker (Fraser, A., Wheeler, P., Cook, P.D. and Sanghvi, Y. S., J. Heterocycl. Chem., 1993, 30, 1277-1287). Itwas observed that the 2′-S-methyl substitution in deoxynucleosideconfers more conformational rigidity to the sugar conformation whencompared with deoxyribonucleosides.

Structural features of DNA:RNA, OMe-DNA:RNA and SMe-DNA:RNA hybrids werealso observed. The average RMS deviation of the DNA:RNA structure fromthe starting hybrid coordinates indicate the structure is stabilizedover the length of the simulation with an approximate average RMSdeviation of 1.0 Å. This deviation is due, in part, to inherentdifferences in averaged structures (i.e. the starting conformation) andstructures at thermal equilibrium. The changes in sugar puckerconformation for three of the central base pairs of this hybrid are ingood agreement with the observations made in previous NMR studies. Thesugars in the RNA strand maintain very stable geometries in the C3′-endoconformation with ring pucker values near 0°. In contrast, the sugars ofthe DNA strand show significant variability.

The average RMS deviation of the OMe-DNA:RNA is approximately 1.2 Å fromthe starting A-form conformation; while the SMe-DNA:RNA shows a slightlyhigher deviation (approximately 1.8 Å) from the starting hybridconformation. The SMe-DNA strand also shows a greater variance in RMSdeviation, suggesting the S-methyl group may induce some structuralfluctuations. The sugar puckers of the RNA complements maintain C3′-endopuckering throughout the simulation. As expected from the nucleosidecalculations, however, significant differences are noted in thepuckering of the OMe-DNA and SMe-DNA strands, with the former adoptingC3′-endo, and the latter, C1′-exo/C2′-endo conformations.

An analysis of the helicoidal parameters for all three hybrid structureshas also been performed to further characterize the duplex conformation.Three of the more important axis-basepair parameters that distinguishthe different forms of the duplexes, X-displacement, propeller twist,and inclination, are reported in Table 10. Usually, an X-displacementnear zero represents a B-form duplex; while a negative displacement,which is a direct measure of deviation of the helix from the helicalaxis, makes the structure appear more A-like in conformation. In A-formduplexes, these values typically vary from −4 Å to −5 Å. In comparingthese values for all three hybrids, the SMe_DNA:RNA hybrid shows themost deviation from the A-form value, the OMe_DNA:RNA shows the least,and the DNA:RNA is intermediate. A similar trend is also evident whencomparing the inclination and propeller twist values with ideal A-formparameters. These results are further supported by an analysis of thebackbone and glycosidic torsion angles of the hybrid structures.Glycosidic angles (X) of A-form geometries, for example, are typicallynear −159° while B form values are near −102°. These angles are found tobe −162°, −133°, and −108° for the OMe-DNA, DNA, and SMe-DNA strands,respectively. All RNA complements adopt an X angle close to −160°. Inaddition, “crankshaft” transitions were also noted in the backbonetorsions of the central UpU steps of the RNA strand in the SMe-DNA:RNAand DNA;RNA hybrids. Such transitions suggest some local conformationalchanges may occur to relieve a less favorable global conformation. Takenoverall, the results indicate the amount of A-character decreases asOMe-DNA:RNA>DNA:RNA>SMe-DNA:RNA, with the latter two adopting moreintermediate conformations when compared to A- and B-form geometries.

TABLE 10 Average helical parameters derived from the last 500 ps ofsimulation time (canonical A-and B-form values are given for comparison)OMe_ SMe_ Helicoidal B-DNA B-DNA A-DNA DNA: DNA: DNA: Parameter (x-ray)(fibre) (fibre) RNA RNA RNA X-disp 1.2 0.0 −5.3 −4.5 −5.4 −3.5Inclination −2.3 1.5 20.7 11.6 15.1 0.7 Propeller −16.4 −13.3 −7.5 −12.7−15.8 −10.3Stability of C2′-modified DNA:RNA hybrids was determined. Although theoverall stability of the DNA:RNA hybrids depends on several factorsincluding sequence-dependencies and the purine content in the DNA or RNAstrands DNA:RNA hybrids are usually less stable than RNA:RNA duplexesand, in some cases, even less stable than DNA:DNA duplexes. Availableexperimental data attributes the relatively lowered stability of DNA:RNAhybrids largely to its intermediate conformational nature betweenDNA:DNA (B-family) and RNA:RNA (A-family) duplexes. The overallthermodynamic stability of nucleic acid duplexes may originate fromseveral factors including the conformation of backbone, base-pairing andstacking interactions. While it is difficult to ascertain the individualthermodynamic contributions to the overall stabilization of the duplex,it is reasonable to argue that the major factors that promote increasedstability of hybrid duplexes are better stacking interactions(electrostatic π-π interactions) and more favorable groove dimensionsfor hydration. The C2′-S-methyl substitution has been shown todestabilize the hybrid duplex. The notable differences in the risevalues among the three hybrids may offer some explanation. While the2′-S-methyl group has a strong influence on decreasing the base-stackingthrough high rise values (˜3.2 Å), the 2′-O-methyl group makes theoverall structure more compact with a rise value that is equal to thatof A-form duplexes (˜2.6 Å). Despite its overall A-like structuralfeatures, the SMe DNA:RNA hybrid structure possesses an average risevalue of 3.2 Å which is quite close to that of B-family duplexes. Infact, some local base-steps (CG steps) may be observed to have unusuallyhigh rise values (as high as 4.5 Å). Thus, the greater destabilizationof 2′-S-methyl substituted DNA:RNA hybrids may be partly attributed topoor stacking interactions.

It has been postulated that RNase H binds to the minor groove of RNA:DNAhybrid complexes, requiring an intermediate minor groove width betweenideal A- and B-form geometries to optimize interactions between thesugar phosphate backbone atoms and RNase H. A close inspection of theaveraged structures for the hybrid duplexes using computer simulationsreveals significant variation in the minor groove width dimensions asshown in Table 11. Whereas the O-methyl substitution leads to a slightexpansion of the minor groove width when compared to the standardDNA:RNA complex, the S-methyl substitution leads to a generalcontraction (approximately 0.9 Å). These changes are most likely due tothe preferred sugar puckering noted for the antisense strands whichinduce either A- or B-like single strand conformations. In addition tominor groove variations, the results also point to potential differencesin the steric makeup of the minor groove. The O-methyl group points intothe minor groove while the S-methyl is directed away towards the majorgroove. Essentially, the S-methyl group has flipped through the basesinto the major groove as a consequence of C2′-endo puckering.

TABLE 11 Minor groove widths averaged over the last 500 ps of simulationtime Phosphate DNA: OMe_DNA: SMe_DNA: DNA: RNA RNA: RNA Distance RNA RNARNA (B-form) (A-form) P5-P20 15.27 16.82 13.73 14.19 17.32 P6-P19 15.5216.79 15.73 12.66 17.12 P7-P18 15.19 16.40 14.08 11.10 16.60 P8-P1715.07 16.12 14.00 10.98 16.14 P9-P16 15.29 16.25 14.98 11.65 16.93P10-P15 15.37 16.57 13.92 14.05 17.69

In addition to the modifications described above, the nucleotides of thechimeric oligomeric compounds of the invention can have a variety ofother modification so long as these other modifications do notsignificantly detract from the properties described above. Thus, fornucleotides that are incorporated into oligonucleotides of theinvention, these nucleotides can have sugar portions that correspond tonaturally-occurring sugars or modified sugars. Representative modifiedsugars include carbocyclic or acyclic sugars, sugars having substituentgroups at their 2′ position, sugars having substituent groups at their3′ position, and sugars having substituents in place of one or morehydrogen atoms of the sugar. Other altered base moieties and alteredsugar moieties are disclosed in U.S. Pat. No. 3,687,808 and PCTapplication PCT/US89/02323.

2′-Endo Regions

A number of different nucleosides can be used independently orexclusively to create one or more of the C2′-endo regions to preparechimeric oligomeric compounds of the present invention. For the purposeof the present invention the terms 2′-endo and C2′-endo are meant toinclude 04′-endo and 2′-deoxy nucleosides. 2′-Deoxy nucleic acids preferboth C2′-endo sugar pucker and O4′-endo sugar, i.e., also known asSouthern pucker, which is thought to impart a less stable B-formgeometry (Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y. and Berger, et. al., Nucleic AcidsResearch, 1998, 26, 2473-2480). The 2′-deoxyribonucleoside is onesuitable nucleoside for the 2′-endo regions but all manner ofnucleosides known in the art that have a preference for 2′-endo sugarconformational geometry are amenable to the present invention. Suchnucleosides include without limitation 2′-modified ribonucleosides suchas for example: 2′-SCH₃, 2′-NH₂, 2′-NH(C₁-C₂ alkyl), 2′-N(C₁-C₂ alkyl)₂,2′-CF₃, 2′=CH₂, 2′=CHF, 2′=CF₂, 2′-CH₃, 2′-C₂H₅, 2′-CH═CH₂ or 2′-C≡CH.Also amenable to the present invention are modified2′-arabinonucleosides including without limitation: 2′-CN, 2′-F, 2′-Cl,2′-Br, 2′-N₃ (azido), 2′-OH, 2′-O—CH₃ or 2′-dehydro-2′-CH₃.

Suitable sugar modifications for the 2′-endo regions of the presentinvention include without limitation 2′-deoxy-2′-S-methyl,2′-deoxy-2′-methyl, 2′-deoxy-2′-amino, 2′-deoxy-2′-mono or dialkylsubstituted amino, 2′-deoxy-2′-fluoromethyl, 2′-deoxy-2′-difluoromethyl,2′-deoxy-2′-trifluoromethyl, 2′-deoxy-2′-methylene,2′-deoxy-2′-fluoromethylene, 2′-deoxy-2′-difluoromethylene,2′-deoxy-2′-ethyl, 2′-deoxy-2′-ethylene and 2′-deoxy-2′-acetylene. Thesenucleotides can alternately be described as 2′-SCH₃ ribonucleotide,2′-CH₃ ribonucleotide, 2′—NH₂ ribonucleotide 2′—NH(C₁-C₂ alkyl)ribonucleotide, 2′-N(C₁-C₂ alkyl)₂ ribonucleotide, 2′-CH₂Fribonucleotide, 2′-CHF₂ ribonucleotide, 2′-CF₃ ribonucleotide, 2′=CH₂ribonucleotide, 2′=CHF ribonucleotide, 2′=CF₂ ribonucleotide, 2′-C₂H₅ribonucleotide, 2′-CH═CH₂ ribonucleotide, 2′-C□CH ribonucleotide. Afurther useful sugar modification is one having a ring located on theribose ring in a cage-like structure including3′,O,4′-C-methyleneribonucleotides. Such cage-like structures willphysically fix the ribose ring in the desired conformation.

Additionally, suitable sugar modifications for the 2′-endo regions ofthe present invention include without limitation are arabino nucleotideshaving 2′-deoxy-2′-cyano, 2′-deoxy-2′-fluoro, 2′-deoxy-2′-chloro,2′-deoxy-2′-bromo, 2′-deoxy-2′-azido, 2′-methoxy and the unmodifiedarabino nucleotide (that includes a 2′-OH projecting upwards towards thebase of the nucleotide). These arabino nucleotides can alternately bedescribed as 2′-CN arabino nucleotide, 2′-F arabino nucleotide, 2′-C1arabino nucleotide, 2′-Br arabino nucleotide, 2′-N₃ arabino nucleotide,2′-O—CH₃ arabino nucleotide and arabino nucleotide.

Such nucleotides are linked together via phosphorothioate,phosphorodithioate, boranophosphate or phosphodiester linkages.Particularly suitable is the phosphorothioate linkage.

Internucleoside Linkages

Specific examples of chimeric oligomeric compounds useful in thisinvention include oligonucleotides containing modified e.g.non-naturally occurring internucleoside linkages. As defined in thisspecification, oligonucleotides having modified internucleoside linkagesinclude internucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.Modified internucleoside linkages containing a phosphorus atom thereininclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Oligonucleotides having inverted polarity comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, each of which is herein incorporated by reference.

In other embodiments of the invention, chimeric oligomeric compoundsinclude one or more phosphorothioate and/or heteroatom internucleosidelinkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—]. The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Amideinternucleoside linkages are disclosed in the above referenced U.S. Pat.No. 5,602,240.

Modified internucleoside linkages that do not include a phosphorus atomtherein include those formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

Conjugate Groups

An additional substitution that can be appended to the oligomericcompounds of the invention involves the linkage of one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the resulting oligomeric compounds. In one embodimentsuch modified oligomeric compounds are prepared by covalently attachingconjugate groups to functional groups such as hydroxyl or amino groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmaco-kinetic properties of oligomers.Typical conjugates groups include cholesterols, lipids, phospho-lipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve oligomer uptake, enhance oligomer resistance todegradation, and/or strengthen sequence-specific hybridization with RNA.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve oligomer uptake,distribution, metabolism or excretion. Representative conjugate groupsare disclosed in International Patent Application PCT/US92/09196, filedOct. 23, 1992 the entire disclosure of which is incorporated herein byreference. Conjugate moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids

Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

The chimeric oligomeric compounds of the invention may also beconjugated to active drug substances, for example, aspirin, warfarin,phenylbutazone, ibuprofen, naproxen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 which is incorporated herein byreference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

Oligomeric compounds used in the compositions of the present inventioncan also be modified to have one or more stabilizing groups that aregenerally attached to one or both termini of oligomeric compounds toenhance properties such as for example nuclease stability. Included instabilizing groups are cap structures. By “cap structure or terminal capmoiety” is meant chemical modifications, which have been incorporated ateither terminus of oligonucleotides (see for example Wincott et al., WO97/26270, incorporated by reference herein). These terminalmodifications protect the oligomeric compounds having terminal nucleicacid molecules from exonuclease degradation, and can help in deliveryand/or localization within a cell. The cap can be present at the5′-terminus (5′-cap) or at the 3′-terminus (3′-cap) or can be present onboth termini. In non-limiting examples, the 5′-cap includes invertedabasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl)nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl riucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270, which isincorporated by reference herein.

Particularly suitable 3′-cap structures of the present inventioninclude, for example 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide, carbocyclicnucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate,3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecylphosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide;L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Tyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

Further 3′ and 5′-stabilizing groups that can be used to cap one or bothends of an oligomeric compound to impart nuclease stability includethose disclosed in WO 03/004602.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound”refers to a polymeric structure capable of hybridizing a region of anucleic acid molecule. This term includes oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics andcombinations of these. Oligomeric compounds routinely prepared linearlybut can be joined or otherwise prepared to be circular and may alsoinclude branching. Oligomeric compounds can hybridized to form doublestranded compounds which can be blunt ended or may include overhangs. Ingeneral an oligomeric compound comprises a backbone of linked momericsubunits where each linked momeric subunit is directly or indirectlyattached to a heterocyclic base moiety. The linkages joining themonomeric subunits, the sugar moieties or surrogates and theheterocyclic base moieties can be independently modified giving rise toa plurality of motifs for the resulting oligomeric compounds includinghemimers, gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety ofthe sugar. In forming oligonucleotides, the phosphate groups covalentlylink adjacent nucleosides to one another to form a linear polymericcompound. The respective ends of this linear polymeric structure can bejoined to form a circular structure by hybridization or by formation ofa covalent bond, however, open linear structures are generally suitable.Within the oligonucleotide structure, the phosphate groups are commonlyreferred to as forming the internucleoside linkages of theoligonucleotide. The normal internucleoside linkage of RNA and DNA is a3′ to 5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA). This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent internucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring portions which function ina similar manner to oligonulceotides. Such non-naturally occurringoligonucleotides are often desired, the naturally occurring formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers tonucleosides that are joined by internucleoside linkages that do not havephosphorus atoms. Internucleoside linkages of this type include shortchain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatomcycloalkyl, one or more short chain heteroatomic and one or more shortchain heterocyclic. These internucleoside linkages include but are notlimited to siloxane, sulfide, sulfoxide, sulfone, acetyl, formacetyl,thioformacetyl, methylene formacetyl, thioformacetyl, alkeneyl,sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide,amide and others having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

Further included in the present invention are oligomeric compounds suchas antisense oligomeric compounds, antisense oligonucleotides, alternatesplicers and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these oligomeric compoundsmay be introduced in the form of single-stranded, double-stranded,circular or hairpin oligomeric compounds and may contain structuralelements such as internal or terminal bulges or loops or mismatches.Once introduced to a system, the oligomeric compounds of the inventionmay elicit the action of one or more enzymes or structural proteins toeffect modification of the target nucleic acid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense oligomeric compoundswhich are “DNA-like” or have “DNA-like” regions elicit RNAse H.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of oligonucleotide-mediatedinhibition of gene expression. Similar roles have been postulated forother ribonucleases such as those in the RNase III and ribonuclease Lfamily of enzymes.

While one form of antisense acting chimeric oligomeric compound is asingle-stranded chimeric oligonucleotide, in many species theintroduction of double-stranded structures, such as double-stranded RNA(dsRNA) molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon, which has been designated RNAinterference (RNAi), occurs in both plants and animals and is believedto have an evolutionary connection to viral defense and transposonsilencing. The term RNAi has been generalized to mean antisense-mediatedgene silencing involving the introduction of dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels (Fire etal., Nature, 1998, 391, 806-811). It has been shown that it is, in fact,the single-stranded RNA oligomers of antisense polarity of the dsRNAswhich are the potent inducers of RNAi (Tijsterman et al., Science, 2002,295, 694-697). The primary interference effects of dsRNAs areposttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA,1998, 95, 15502-15507).

In addition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodifications. These modifications either alone or in combination withother nucleosides may enhance one or more of the desired propertiesdescribed above. Thus, for nucleotides that are incorporated intooligonucleotides of the invention, these nucleotides can have sugarportions that correspond to naturally-occurring sugars or modifiedsugars. Representative modified sugars include carbocyclic or acyclicsugars, sugars having substituent groups at one or more of their 2′, 3′or 4′ positions and sugars having substituents in place of one or morehydrogen atoms of the sugar. Additional nucleosides amenable to thepresent invention having altered base moieties and or altered sugarmoieties are disclosed in U.S. Pat. No. 3,687,808 and PCT applicationPCT/US89/02323.

The oligomeric compounds in accordance with this invention comprise fromabout 5 to about 80 nucleobases (i.e. from about 5 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies oligomeric compounds of 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases inlength, or any sub-range therewithin.

In a further embodiment, the oligomeric compounds of the invention are 5to 50 nucleobases in length. One of ordinary skill in the art willappreciate that the invention embodies oligomeric compounds of 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49 or 50 nucleobases in length, or any sub-rangetherewithin.

In another embodiment, the oligomeric compounds of the invention are 12to 50 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobasesin length, or any sub-range therewithin.

In another embodiment, the oligomeric compounds of the invention are 12to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50nucleobases in length, or any sub-range therewithin.

In a further embodiment, the oligomeric compounds of the invention are13 to 40 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39 or 40 nucleobases in length, or any sub-rangetherewithin.

In another embodiment, the oligomeric compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length,or any sub-range therewithin.

In another embodiment, the oligomeric compounds of the invention are 15to 25 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies oligomeric compounds of 15, 16, 17, 18,19, 20, 21, 22, 23, 24 or 25 nucleobases in length, or any sub-rangetherewithin.

In a further embodiment, the oligomeric compounds of the invention are21 to 25 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 21, 22, 23,24 or 25 nucleobases in length, or any sub-range therewithin.

Particularly suitable oligomeric compounds are oligonucleotidescomprising from about 12 to about 50 nucleobases, from about 13 to 40nucleobases, or from about 15 to about 30 nucleobases.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications ofChemically synthesized RNA in RNA:Protein Interactions, Ed. Smith(1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713) synthesisas appropriate. In addition specific protocols for the synthesis ofoligomeric compounds of the invention are illustrated in the examplesbelow.

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The present invention is also useful for the preparation of oligomericcompounds incorporating at least one 2′-O-protected nucleoside. Afterincorporation and appropriate deprotection the 2′-O-protected nucleosidewill be converted to a ribonucleoside at the position of incorporation.The number and position of the 2-ribonucleoside units in the finaloligomeric compound can vary from one at any site or the strategy can beused to prepare up to a full 2′-OH modified oligomeric compound. All2′-O-protecting groups amenable to the synthesis of oligomeric compoundsare included in the present invention. In general a protected nucleosideis attached to a solid support by for example a succinate linker. Thenthe oligonucleotide is elongated by repeated cycles of deprotecting the5′-terminal hydroxyl group, coupling of a further nucleoside unit,capping and oxidation (alternatively sulfurization). In a morefrequently used method of synthesis the completed oligonucleotide iscleaved from the solid support with the removal of phosphate protectinggroups and exocyclic amino protecting groups by treatment with anammonia solution. Then a further deprotection step is normally requiredfor the more specialized protecting groups used for the protection of2′-hydroxyl groups which will give the fully deprotectedoligonucleotide.

A large number of 2′-O-protecting groups have been used for thesynthesis of oligoribo-nucleotides but over the years more effectivegroups have been discovered. The key to an effective 2′-O-protectinggroup is that it is capable of selectively being introduced at the2′-O-position and that it can be removed easily after synthesis withoutthe formation of unwanted side products. The protecting group also needsto be inert to the normal deprotecting, coupling, and capping stepsrequired for oligoribonucleotide synthesis. Some of the protectinggroups used initially for oligoribonucleotide synthesis includedtetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two groupsare not compatible with all 5′-O-protecting groups so modified versionswere used with 5′-DMT groups such as1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp). Reese has identifieda number of piperidine derivatives (like Fpmp) that are useful in thesynthesis of oligoribonucleotides including1-[(chloro-4-methyl)phenyl]-4′-methoxypiperidin-4-yl (Reese et al.,Tetrahedron Lett., 1986, (27), 2291). Another approach was to replacethe standard 5′-DMT (dimethoxytrityl) group with protecting groups thatwere removed under non-acidic conditions such as levulinyl and9-fluorenylmethoxycarbonyl. Such groups enable the use of acid labile2′-protecting groups for oligoribonucleotide synthesis. Another morewidely used protecting group initially used for the synthesis ofoligoribonucleotides was the t-butyldimethylsilyl group (Ogilvie et al.,Tetrahedron Lett., 1974, 2861; Hakimelahi et al., Tetrahedron Lett.,1981, (22), 2543; and Jones et al., J. Chem. Soc. Perkin I, 2762). The2′-O-protecting groups can require special reagents for their removalsuch as for example the t-butyldimethylsilyl group is normally removedafter all other cleaving/deprotecting steps by treatment of theoligomeric compound with tetrabutylammonium fluoride (TBAF).

One group of researchers examined a number of 2′-O-protecting groups(Pitsch, S., Chimia, 2001, (55), 320-324.) The group examined fluoridelabile and photolabile protecting groups that are removed using moderateconditions. One photolabile group that was examined was the[2-(nitrobenzyl)oxy]methyl (nbm) protecting group (Schwartz et al.,Bioorg. Med. Chem. Lett., 1992, (2), 1019.) Other groups examinedincluded a number structurally related formaldehyde acetal-derived,2′-O-protecting groups. Also prepared were a number of relatedprotecting groups for preparing 2′-O-alkylated nucleosidephosphoramidites including 2′-O—[(triisopropylsilyl)oxy]methyl(2′-O—CH₂—O—Si(iPr)₃, TOM). One 2′-O-protecting group that was preparedto be used orthogonally to the TOM group was2′-O—[(R)-1-(2-nitrophenyl)ethyloxy)methyl] ((R)-mnbm).

Another strategy using a fluoride labile 5′-O-protecting group (non-acidlabile) and an acid labile 2′-O-protecting group has been reported(Scaringe, Stephen A., Methods, 2001, (23) 206-217). A number ofpossible silyl ethers were examined for 5′-O-protection and a number ofacetals and orthoesters were examined for 2′-O-protection. Theprotection scheme that gave the best results was 5′-O-silyl ether-2′-ACE(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether(DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses amodified phosphoramidite synthesis approach in that some differentreagents are required that are not routinely used for RNA/DNA synthesis.

Although a lot of research has focused on the synthesis ofoligoribonucleotides the main RNA synthesis strategies that arepresently being used commercially include5′-O-DMT-2′-O-t-butyldimethylsilyl (TBDMS), 5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl] (FPMP),2′-O-[(triisopropylsilyl)oxy]methyl (2′-O—CH₂—O—Si(iPr)₃ (TOM), and the5′-O-silyl ether-2′-ACE (5′-O-bis(trimethylsiloxy)cyclododecyloxysilylether (DOD)-2′-O-bis(2-acetoxyethoxy)methyl (ACE). A current list ofsome of the major companies currently offering RNA products includePierce Nucleic Acid Technologies, Dharmacon Research Inc., AmeriBiotechnologies Inc., and Integrated DNA Technologies, Inc. One company,Princeton Separations, is marketing an RNA synthesis activatoradvertised to reduce coupling times especially with TOM and TBDMSchemistries. Such an activator would also be amenable to the presentinvention.

The primary groups being used for commercial RNA synthesis are:

-   -   TBDMS=5′-O-DMT-2′-O-t-butyldimethylsilyl;    -   TOM=2′-O-[(triisopropylsilyl)oxy]methyl;    -   DOD/ACE=(5′-O-bis(trimethylsiloxy)cyclododecyloxysilyl        ether-2′-O-bis(2-acetoxyethoxy)methyl    -   FPMP=5′-O-DMT-2′-O-[1(2-fluorophenyl)-4-methoxypiperidin-4-yl].

All of the aforementioned RNA synthesis strategies are amenable to thepresent invention. Strategies that would be a hybrid of the above e.g.using a 5′-protecting group from one strategy with a 2′-O-protectingfrom another strategy is also amenable to the present invention.

The preparation of ribonucleotides and oligomeric compounds having atleast one ribonucleoside incorporated and all the possibleconfigurations falling in between these two extremes are encompassed bythe present invention. The corresponding oligomeric compounds can behybridized to further oligomeric compounds includingoligoribonucleotides having regions of complementarity to formdouble-stranded (duplexed) oligomeric compounds, which are commonlyreferred to as dsRNAs in the art. Such double stranded oligonucleotidemoieties have been shown in the art to modulate target expression andregulate translation as well as RNA processsing via an antisensemechanism. Moreover, the double-stranded moieties may be subject tochemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmonsand Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263,103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al.,Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., GenesDev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498;Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, suchdouble-stranded moieties have been shown to inhibit the target by theclassical hybridization of antisense strand of the duplex to the target,thereby triggering enzymatic degradation of the target (Tijsterman etal., Science, 2002, 295, 694-697). The effects of nucleosidemodifications on RNAi activity are evaluated according to existingliterature (Elbashir et al., Nature (2001), 411, 494-498; Nishikura etal., Cell (2001), 107, 415-416; and Bass et al., Cell (2000), 101,235-238.)

The methods of preparing oligomeric compounds of the present inventioncan also be applied in the areas of drug discovery and targetvalidation.

Oligomer Mimetics (Oligonucleotide Mimics)

Another group of oligomeric compounds amenable to the present inventionincludes oligonucleotide mimetics. The term mimetic as it is applied tooligonucleotides is intended to include oligomeric compounds whereinonly the furanose ring or both the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.One such oligomeric compound, an oligonucleotide mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA oligomeric compounds, thesugar-backbone of an oligonucleotide is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA oligomericcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA oligomeric compounds can be found inNielsen et al., Science, 1991, 254, 1497-1500.

PNA has been modified in the art to incorporate numerous modificationssince the basic PNA structure was first prepared. The basic structure isshown below:

wherein

Bx is a heterocyclic base moiety;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₂ alkyl, substituted or unsubstituted C₂-C₁₂ alkenyl,substituted or unsubstituted C₂-C₁₂ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the ω-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the ω-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the ω-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 2 to about 50.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. One class of linking groups has been selected to give anon-ionic oligomeric compound. The non-ionic morpholino-based oligomericcompounds are less likely to have undesired interactions with cellularproteins. Morpholino-based oligomeric compounds are non-ionic mimics ofoligonucleotides which are less likely to form undesired interactionswith cellular proteins (Dwaine A. Braasch and David R. Corey,Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based oligomericcompounds are disclosed in U.S. Pat. No. 5,034,506, issued Jul. 23,1991. The morpholino class of oligomeric compounds has been preparedhaving a variety of different linking groups joining the monomericsubunits.

Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

wherein

T₁ is hydroxyl or a protected hydroxyl;

T₅ is hydrogen or a phosphate or phosphate derivative;

L₂ is a linking group; and

n is from 2 to about 50.

A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present inan DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligonucleotideshaving specific positions modified with CeNA have been prepared andstudied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). Ingeneral the incorporation of CeNA monomers into a DNA chain increasesits stability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichroism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. coli RNase resulting in cleavage of the targetRNA strand.

The general formula of CeNA is shown below:

wherein

each Bx is a heterocyclic base moiety;

T₁ is hydroxyl or a protected hydroxyl; and

T2 is hydroxyl or a protected hydroxyl.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid)can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

Another group of modifications includes nucleosides having sugarmoieties that are bicyclic thereby locking the sugar conformationalgeometry. The most studied of these nucleosides having a bicyclic sugarmoiety is locked nucleic acid or LNA. As can be seen in the structurebelow the 2′-O-has been linked via a methylene group to the 4′ carbon.This bridge attaches under the 3′ bonds forcing the sugar ring into alocked 3′-endo conformation geometry. The linkage can be a methylene(—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atomwherein n is 1 for LNA. LNA and LNA analogs display very high duplexthermal stabilities with complementary DNA and RNA (T_(m)=+3 to +10 C),stability towards 3′-exonucleolytic degradation and good solubilityproperties.

An LNA analog that also has been looked at is ENA wherein an additionalmethylene group has been added to the bridge between the 2′ and the 2′carbons (4′-CH₂—CH₂—O-2′, Kaneko et al., United States PatentApplication Publication No.: US 2002/0147332, Singh et al., Chem.Commun., 1998, 4, 455-456, also see Japanese Patent ApplicationHEI-11-33863, Feb. 12, 1999).

In another publication a large genus of nucleosides having bicyclicsugar moieties is disclosed. The bridging group is variable as are thepoints of attachment (United States Patent Application Publication No.:U.S. 2002/0068708).

The basic structure of LNA showing the bicyclic ring system is shownbelow:

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (T_(m)=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.

Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

Potent and nontoxic antisense oligonucleotides containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97,5633-5638.) The authors have demonstrated that LNAs confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

One group has added an additional methylene group to the LNA2′,4′-bridging group (e.g. 4′-CH₂—CH₂—O-2′ (ENA), Kaneko et al., UnitedStates Patent Application Publication No.: US 2002/0147332, also seeJapanese Patent Application HEI-11-33863, Feb. 12, 1999).

Further oligonucleotide mimetics have been prepared to include bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., J.Am. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogshave been oligomerized using the phosphoramidite approach and theresulting oligomeric compounds containing tricyclic nucleoside analogshave shown increased thermal stabilities (T_(m)'s) when hybridized toDNA, RNA and itself. Oligomeric compounds containing bicyclic nucleosideanalogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in abackbone the backbone. This class of olignucleotide mimetic is reportedto have useful physical and biological and pharmacological properties inthe areas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of Markush variables see: U.S. Pat.Nos. 5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Modified Sugars

Oligomeric compounds of the invention may also contain one or moresubstituted sugar moieties. Oligomeric compounds comprise a sugarsubstituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₂ alkylor C₂ to C₁₂ alkenyl and alkynyl. Particularly suitable areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other oligonucleotides comprise a sugar substituent groupselected from: C₁ to C₁₂ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Onemodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995,78, 486-504) i.e., an alkoxyalkoxy group. Another modification includes2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂.

Other sugar substituent groups include methoxy (—O—CH₃), aminopropoxy(—OCH₂CH₂CH₂NH₂), allyl (—CH₂—CH═CH₂), —O-allyl (—O—CH₂—CH═CH₂) andfluoro (F). 2′-Sugar substituent groups may be in the arabino (up)position or ribo (down) position. One 2′-arabino modification is 2′-F.Similar modifications may also be made at other positions on theoligomeric compound, particularly the 3′ position of the sugar on the 3′terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligomeric compounds may also havesugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative United States patents that teachthe preparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, each of whichis herein incorporated by reference in its entirety.

Further representative sugar substituent groups include groups offormula I_(a) or II_(a):

wherein:

R_(b) is O, S or NH;

R_(d) is a single bond, O, S or C(═O);

R_(e) is C₁-C₁₂ alkyl, N(R_(k))(R_(m)), N(R_(k))(R_(n)),N═C(R_(p))(R_(q)), N═C(R_(p))(R_(r)) or has formula III_(a);

R_(p) and R_(q) are each independently hydrogen or C₁-C₁₂ alkyl;

R_(r) is —R_(x)—R_(y);

each R_(s), R_(t), R_(u) and R_(v) is, independently, hydrogen,C(O)R_(w), substituted or unsubstituted C₁-C₁₂ alkyl, substituted orunsubstituted C₂-C₁₂ alkenyl, substituted or unsubstituted C₂-C₁₂alkynyl, alkylsulfonyl, arylsulfonyl, a chemical functional group or aconjugate group, wherein the substituent groups are selected fromhydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol,thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl;

or optionally, R_(u) and R_(v), together form a phthalimido moiety withthe nitrogen atom to which they are attached;

each R_(w) is, independently, substituted or unsubstituted C₁-C₁₂alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl;

R_(k) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

R_(p) is hydrogen, a nitrogen protecting group or —R_(x)—R_(y);

R_(x) is a bond or a linking moiety;

R_(y) is a chemical functional group, a conjugate group or a solidsupport medium;

each R_(m) and R_(n) is, independently, H, a nitrogen protecting group,substituted or unsubstituted C₁-C₁₂ alkyl, substituted or unsubstitutedC₂-C₁₂ alkenyl, substituted or unsubstituted C₂-C₁₂ alkynyl, wherein thesubstituent groups are selected from hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl,alkynyl; NH₃ ⁺, N(R_(u))(R_(v)), guanidino and acyl where said acyl isan acid amide or an ester;

or R_(m) and R_(n), together, are a nitrogen protecting group, arejoined in a ring structure that optionally includes an additionalheteroatom selected from N and O or are a chemical functional group;

R_(i) is OR_(z), SR_(z), or N(R_(z))₂;

each R_(z) is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,C(═NH)N(H)R_(u), C(═O)N(H)R_(u) or OC(═O)N(H)R_(u);

R_(f), R_(g) and R_(h) comprise a ring system having from about 4 toabout 7 carbon atoms or having from about 3 to about 6 carbon atoms and1 or 2 heteroatoms wherein said heteroatoms are selected from oxygen,nitrogen and sulfur and wherein said ring system is aliphatic,unsaturated aliphatic, aromatic, or saturated or unsaturatedheterocyclic;

R_(j) is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(R_(k))(R_(m))OR_(k),halo, SR_(k) or CN;

m_(a) is 1 to about 10;

each mb is, independently, 0 or 1;

mc is 0 or an integer from 1 to 10;

md is an integer from 1 to 10;

me is from 0, 1 or 2; and

provided that when mc is 0, md is greater than 1.

Representative substituents groups of Formula Ia are disclosed in U.S.patent application Ser. No. 09/130,973, filed Aug. 7, 1998, entitled“Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by referencein its entirety.

Representative cyclic substituent groups of Formula IIa are disclosed inU.S. patent application Ser. No. 09/123,108, filed Jul. 27, 1998,entitled “RNA Targeted 2′-Oligomeric compounds that are ConformationallyPreorganized,” hereby incorporated by reference in its entirety.

Sugar substituent groups include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)CH₃,O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)NH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.

Representative guanidino substituent groups that are shown in formulaIIIa disclosed in co-owned U.S. patent application Ser. No. 09/349,040,entitled “Functionalized Oligomers”, filed Jul. 7, 1999, herebyincorporated by reference in its entirety.

Representative acetamido substituent groups are disclosed in U.S. Pat.No. 6,147,200 which is hereby incorporated by reference in its entirety.

Representative dimethylaminoethyloxyethyl substituent groups aredisclosed in International Patent Application PCT/US99/17895, entitled“2′-O-Dimethylaminoethyloxyethyl-Oligomeric compounds”, filed Aug. 6,1999, hereby incorporated by reference in its entirety.

Modified Nucleobases/Naturally Occurring Nucleobases

Chimeric oligomeric compounds of the invention may also includenucleobase (often referred to in the art simply as “base” or“heterocyclic base moiety”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases also referred hereinas heterocyclic base moieties include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S.T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently suitable base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

In one aspect of the present invention chimeric oligomeric compounds areprepared having polycyclic heterocyclic compounds in place of one ormore heterocyclic base moieties. A number of tricyclic heterocycliccompounds have been previously reported. These compounds are routinelyused in antisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs. Many of these polycyclic heterocyclic compounds have thegeneral formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═H) (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846), 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁-R₁₄═H), (Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874) and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═F) (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388). Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric oligomeric compounds” filed May 24, 2002,Ser. No. 10/013,295, both of which are herein incorporated by referencein their entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀═O, R₁₁═—O—(CH₂)₂—NH₂, R₁₂₋₁₄═H)(Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532).Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification. On the other hand, the gain in helical stabilitydoes not compromise the specificity of the oligonucleotides. The T_(m)data indicate an even greater discrimination between the perfect matchand mismatched sequences compared to dC5^(me). It was suggested that thetethered amino group serves as an additional hydrogen bond donor tointeract with the Hoogsteen face, namely the O6, of a complementaryguanine thereby forming 4 hydrogen bonds. This means that the increasedaffinity of G-clamp is mediated by the combination of extended basestacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183 and U.S. Pat. No. 6,007,992, each of which is incorporatedherein in its entirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity makes them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity (Lin, K-Y; Matteucci, M. J.Am. Chem. Soc. 1998, 120, 8531-8532). The activity enhancement was evenmore pronounced in case of G-clamp, as a single substitution was shownto significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides (Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518). Nevertheless, to optimizeoligonucleotide design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further modified polycyclic heterocyclic compounds useful asheterocyclcic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and Unites States patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, each of which isherein incorporated by reference.

Activated Phosphorus Groups

The compositions of the present invention illustrate the use ofactivated phosphorus compositions (e.g. compounds having activatedphosphorus-containing substituent groups) in coupling reactions. As usedherein, the term activated phosphorus composition includes monomers andoligomers that have an activated phosphorus-containing substituent groupthat is reactive with a hydroxyl group of another monomeric oroligomeric compound to form a phosphorus-containing internucleotidelinkage. Such activated phosphorus groups contain activated phosphorusatoms in P^(III) valence state and are known in the art and include, butare not limited to, phosphoramidite, H-phosphonate, phosphate triestersand chiral auxiliaries. One synthetic solid phase synthesis utilizesphosphoramidites as activated phosphates. The phosphoramidites utilizeP^(III) chemistry. The intermediate phosphite compounds are subsequentlyoxidized to the P^(V) state using known methods to yield, in oneembodiment, phosphodiester or phosphorothioate internucleotide linkages.Additional activated phosphates and phosphites are disclosed inTetrahedron Report Number 309 (Beaucage and Iyer, Tetrahedron, 1992, 48,2223-2311).

Activated phosphorus groups are useful in the preparation of a widerange of oligomeric compounds including but not limited tooligonucleosides and oligonucleotides as well as oligonucleotides thathave been modified or conjugated with other groups at the base or sugaror both. Also included are oligonucleotide mimetics including but notlimited to peptide nucleic acids (PNA), morpholino nucleic acids,cyclohexenyl nucleic acids (CeNA), anhydrohexitol nucleic acids, lockednucleic acids (LNA and ENA), bicyclic and tricyclic nucleic acids,phosphonomonoester nucleic acids and cyclobutyl nucleic acids. Arepresentative example of one type of oligomer synthesis that utilizesthe coupling of an activated phosphorus group with a reactive hydroxylgroup is the widely used phosphoramidite approach. A phosphoramiditesynthon is reacted under appropriate conditions with a reactive hydroxylgroup to form a phosphite linkage that is further oxidized to aphosphodiester or phosphorothioate linkage. This approach commonlyutilizes nucleoside phosphoramidites of the formula:

whereineach Bx′ is an optionally protected heterocyclic base moiety;each R_(1′) is, independently, H or an optionally protected sugarsubstituent group;T_(3′) is H, a hydroxyl protecting group, a nucleoside, a nucleotide, anoligonucleoside or an oligonucleotide;

L₁ is N(R₁)R₂;

each R₂ and R₃ is, independently, C₁-C₁₂ straight or branched chainalkyl;or R₂ and R₃ are joined together to form a 4- to 7-membered heterocyclicring system including the nitrogen atom to which R₂ and R₃ are attached,wherein said ring system optionally includes at least one additionalheteroatom selected from O, N and S;L₂ is Pg-O—, Pg-S—, C₁-C₁₂ straight or branched chain alkyl,CH₃(CH₂)₀₋₁₀—O— or —NR₅R₆;Pg is a protecting/blocking group; andeach R₅ and R₆ is, independently, hydrogen, C₁-C₁₂ straight or branchedchain alkyl, cycloalkyl or aryl;or optionally, R₅ and R₆, together with the nitrogen atom to which theyare attached form a cyclic moiety that may include an additionalheteroatom selected from O, S and N; or

L₁ and L₂ together with the phosphorus atom to which L₁ and L₂ areattached form a chiral auxiliary.

Groups that are attached to the phosphorus atom of internucleotidelinkages before and after oxidation (L₁ and L₂) can include nitrogencontaining cyclic moieties such as morpholine. Such oxidizedinternucleoside linkages include a phosphoromorpholidothioate linkage(Wilk et al., Nucleosides and nucleotides, 1991, 10, 319-322). Furthercyclic moieties amenable to the present invention include mono-, bi- ortricyclic ring moieties which may be substituted with groups such asoxo, acyl, alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino,amido, azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo,haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro, sulfide,sulfone, sulfonamide, thiol and thioalkoxy. A bicyclic ring structurethat includes nitrogen is phthalimido.

Unless otherwise defined herein, alkyl means C₁-C₁₂, C₁-C₈, or C₁-C₆,straight or (where possible) branched chain aliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, C₁-C₈, orC₁-C₆, straight or (where possible) branched chain aliphatic hydrocarbylcontaining at least one or about 1 to about 3, hetero atoms in thechain, including the terminal portion of the chain. Suitable heteroatomsinclude N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, C₃-C₈, orC₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkenyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, C₂-C₈, or C₂-C₆alkynyl, which may be straight or (where possible) branched hydrocarbylmoiety, which contains at least one carbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon. Thenumber of carbon atoms can vary from 1 to about 12, or from 1 to about6, and the total number of ring members can vary from three to about 15,or from about 3 to about 8. Ring heteroatoms can be N, O and S.Heterocycloalkyl groups include morpholino, thiomorpholino, piperidinyl,piperazinyl, homopiperidinyl, homopiperazinyl, homomorpholino,homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl,tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl,tetrahydropyrrazolyl, furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Ayl rings can have about 6to about 20 ring carbons. Aryl rings can include phenyl, napthyl,anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. The ring system can contain about 1 to about 4 rings.The number of carbon atoms can vary from 1 to about 12, or from 1 toabout 6, and the total number of ring members can vary from three toabout 15, or from about 3 to about 8. Ring heteroatoms are N, O and S.Hetaryl moieties include pyrazolyl, thiophenyl, pyridyl, imidazolyl,tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl,benzimidazolyl, benzothiophenyl, etc.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached. Other electronwithdrawing groups of note include those whose electronegativitiesexceed that of carbon, for example halogen, nitro, or phenyl substitutedin the ortho- or para-position with one or more cyano, isothiocyanato,nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings Halo (halogen) substituents can be Cl, Br, and I.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc.

In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate.

Phosphate protecting groups include those described in U.S. Pat. No.5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No. 6,051,699, U.S. Pat.No. 6,020,475, U.S. Pat. No. 6,326,478, U.S. Pat. No. 6,169,177, U.S.Pat. No. 6,121,437, U.S. Pat. No. 6,465,628 each of which is expresslyincorporated herein by reference in its entirety.

Hybridization

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,one mechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases) of thestrands of oligomeric compounds. For example, adenine and thymine arecomplementary nucleobases which pair through the formation of hydrogenbonds. Hybridization can occur under varying circumstances.

An oligomeric compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a reduction in activity, and thereis a sufficient degree of complementarity to avoid off-target effects(non-specific binding of the antisense oligomeric compound to non-targetnucleic acid sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, or under conditions in which assays areperformed in the case of in vitro assays).

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which an oligomericcompound of the invention will hybridize to its target sequence, but toa minimal number of other sequences. Stringent conditions aresequence-dependent and will vary with different circumstances and in thecontext of this invention, “stringent conditions” under which oligomericcompounds hybridize to a target sequence are determined by the natureand composition of the oligomeric compounds and the assays in which theyare being investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases regardless of where the two are located. Forexample, if a nucleobase at a certain position of an oligomeric compoundis capable of hydrogen bonding (pairing) with a nucleobase at a certainposition of a target nucleic acid, the target nucleic acid being a DNA,RNA, or oligonucleotide molecule, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe a complementary position. The oligomeric compound and the furtherDNA, RNA, or oligonucleotide molecule are complementary to each otherwhen a sufficient number of complementary positions in each molecule areoccupied by nucleobases which can hydrogen bond with each other. Thus,“specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of precise pairing or complementarityover a sufficient number of nucleobases such that stable and specificbinding occurs between the oligonucleotide and a target nucleic acid.

It is understood in the art that the sequence of a chimeric oligomericcompound compound need not be 100% complementary to that of its targetnucleic acid to be specifically hybridizable. Moreover, anoligonucleotide may hybridize over one or more segments such thatintervening or adjacent segments are not involved in the hybridizationevent (e.g., a loop structure or hairpin structure). It may be desirablethat the chimeric oligomeric compounds of the present invention compriseat least 70%, at least 80%, at least 90%, at least 95%, or at least 99%sequence complementarity to a target region within the target nucleicacid to which they are targeted. For example, a chimeric oligomericcompound in which 18 of 20 nucleobases are complementary (the remaining2 being mismatches) to a target region, which specifically hybridizes,would represent 90 percent complementarity. In this example, theremaining noncomplementary nucleobases may be clustered or interspersedwith complementary nucleobases and need not be contiguous to each otheror to complementary nucleobases. As such, a chimeric oligomeric compoundwhich is 18 nucleobases in length having 4 (four) noncomplementarynucleobases which are flanked by two regions of complete complementaritywith the target nucleic acid would have 77.8% overall complementaritywith the target nucleic acid and would thus fall within the scope of thepresent invention. Percent complementarity of a chimeric oligomericcompound with a region of a target nucleic acid can be determinedroutinely using BLAST programs (basic local alignment search tools) andPowerBLAST programs known in the art (Altschul et al., J. Mol. Biol.,1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

Targets of the Invention

The chimeric oligomeric compounds of the present invention are targetedto nucleic acid targets in a sequence dependent manner. One nucleic acidtarget is messenger RNA. More specifically, chimeric oligomericcompounds of the invention will modulate gene expression by hybridizingto a nucleic acid target resulting in alteration of or reduction innormal function of the target nucleic acid. As used herein, the term“target nucleic acid” or “nucleic acid target” is used for convenienceto encompass any nucleic acid capable of being targeted includingwithout limitation DNA, RNA (including pre-mRNA and mRNA or portionsthereof) transcribed from such DNA, and also cDNA derived from such RNA.In one embodiment of the invention the target nucleic acid is amessenger RNA. The inhibition of the target is typically based uponhydrogen bonding-based hybridization of the chimeric oligomeric compoundstrands or segments such that at least one strand or segment is cleaved,degraded, or otherwise rendered inoperable. In this regard, it ispresently suitable to target specific nucleic acid molecules and theirfunctions for such inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. In the context of the present invention,“modulation” and “modulation of expression” mean either an increase(stimulation) or a decrease (inhibition) in the amount or levels of anucleic acid molecule encoding the gene, e.g., DNA or RNA Inhibition isoften the desired form of modulation of expression and mRNA is often asuitable target nucleic acid.

In one aspect, the present invention is directed to chimeric oligomericcompounds that are prepared having enhanced activity against nucleicacid targets. As used herein the phrase “enhanced activity” can indicateupregulation or downregulation of a system. A target and a mechanism forits modulation is determined. An oligonucleotide is selected having aneffective length and sequence that is complementary to a portion of thetarget sequence. The selected sequence is divided into regions and thenucleosides of each region are modified to enhance the desiredproperties of the respective region. Consideration is also given to the5′ and 3′-termini as there are often advantageous modifications that canbe made to one or more of the terminal nucleosides. Furthermodifications are also considered such as internucleoside linkages,conjugate groups, substitute sugars or bases, substitution of one ormore nucleosides with nucleoside mimetics and any other modificationthat can enhance the selected sequence for its intended target.

“Targeting” a chimeric oligomeric compound of the invention to aparticular nucleic acid molecule, in the context of this invention, canbe a multistep process. The process usually begins with theidentification of a target nucleic acid whose function is to bemodulated. This target nucleic acid may be, for example, a cellular gene(or mRNA transcribed from the gene) whose expression is associated witha particular disorder or disease state, or a nucleic acid molecule froman infectious agent.

The targeting process usually also includes determination of at leastone target region, target segment, or target site within the targetnucleic acid for the antisense interaction to occur (hybridization ofthe chimeric oligomeric compound to its complementary sense target) suchthat the desired effect, e.g., modulation of expression, will result.Within the context of the present invention, the term “target region” isdefined as a portion of the target nucleic acid having at least oneidentifiable structure, function, or characteristic. Within regions oftarget nucleic acids are segments. “Target segments” are defined assmaller or sub-portions of regions within a target nucleic acid. “Targetsites,” as used in the present invention, are defined as positionswithin a target nucleic acid. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes has a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA transcribed from a gene encoding anucleic acid target, regardless of the sequence(s) of such codons. It isalso known in the art that a translation termination codon (or “stopcodon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAGand 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the chimeric oligomeric compounds of thepresent invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, oneregion is the intragenic region encompassing the translation initiationor termination codon of the open reading frame (ORF) of a gene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsosuitable to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence, resulting in exon-exon junctions at thesites where exons are joined. Targeting exon-exon junctions can beuseful in situations where the overproduction of a normal splice productis implicated in disease, or where the overproduction of an aberrantsplice product is implicated in disease. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also suitable target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesknown as “fusion transcripts” are also suitable target sites. It is alsoknown that introns can be effectively targeted using chimeric oligomericcompounds targeted to, for example, DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequences.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso suitable target nucleic acids.

The locations on the target nucleic acid to which the chimericoligomeric compounds hybridize are hereinbelow referred to as “suitabletarget segments.” As used herein the term “suitable target segment” isdefined as at least a 5-nucleobase portion of a target region to whichan active chimeric oligomeric compound of the present invention istargeted. While not wishing to be bound by theory, it is presentlybelieved that these target segments represent portions of the targetnucleic acid which are accessible for hybridization.

Exemplary chimeric oligomeric compounds include at least the 5consecutive nucleobases from the 5′-terminus of a targeted nucleic acide.g. a cellular gene or mRNA transcribed from the gene (the remainingnucleobases being a consecutive stretch of the same oligonucleotidebeginning immediately upstream of the 5′-terminus of the chimericoligomeric compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains fromabout 5 to about 80 nucleobases). Similarly, chimeric oligomericcompounds comprise at least the 5 consecutive nucleobases from the3′-terminus of one of the illustrative chimeric oligomeric compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately downstream of the 3′-terminus ofthe chimeric oligomeric compound which is specifically hybridizable tothe target nucleic acid and continuing until the chimeric oligomericcompound contains from about 5 to about 80 nucleobases). One havingskill in the art armed with the chimeric oligomeric compoundsillustrated herein will be able, without undue experimentation, toidentify further chimeric oligomeric compounds.

Once one or more target regions, target segments or target sites havebeen identified, chimeric oligomeric compounds of the invention arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

The oligomeric antisense compounds can also be targeted to regions of atarget nucleobase sequence, such as those disclosed herein. All regionsof a nucleobase sequence to which an oligomeric antisense compound canbe targeted, wherein the regions are greater than or equal to 5 and lessthan or equal to 80 nucleobases, are described as follows:

Let R(m, n+m−1) be a region from a target nucleobase sequence, where “n”is the 5′-most nucleobase position of the region, where “n+m−1” is the3′-most nucleobase position of the region and where “m” is the length ofthe region. A set “S(m)”, of regions of length “m” is defined as theregions where n ranges from 1 to L−m+1, where L is the length of thetarget nucleobase sequence and L>m. A set, “A”, of all regions can beconstructed as a union of the sets of regions for each length from wherem is greater than or equal to 5 and is less than or equal to 80.

This set of regions can be represented using the following mathematicalnotation:

$A = {\bigcup\limits_{m}{S(m)}}$ where  m ∈ N|5 ≤ m ≤ 80 andS(m) = {R_(n, n + m − 1)|n ∈ {1, 2, 3, …  , L − m + 1}}

where the mathematical operator | indicates “such that”,

where the mathematical operator ε indicates “a member of a set” (e.g.yεZ indicates that element y is a member of set Z),

-   -   where x is a variable,

where N indicates all natural numbers, defined as positive integers,

-   -   and where the mathematical operator ∪ indicates “the union of        sets”.

For example, the set of regions for m equal to 5, 20 and 80 can beconstructed in the following manner. The set of regions, each 5nucleobases in length, S(m=5), in a target nucleobase sequence 100nucleobases in length (L=100), beginning at position 1 (n=1) of thetarget nucleobase sequence, can be created using the followingexpression:

S(5)={R _(1,5) |nε{1,2,3, . . . , 96}}

and describes the set of regions comprising nucleobases 1-5, 2-6, 3-7,4-8, 5-9, 6-10, 7-11, 8-12, 9-13, 10-14, 11-15, 12-16, 13-17, 14-18,15-19, 16-20, 17-21, 18-22, 19-23, 20-24, 21-25, 22-26, 23-27, 24-28,25-29, 26-30, 27-31, 28-32, 29-33, 30-34, 31-35, 32-36, 33-37, 34-38,35-39, 36-40, 37-41, 38-42, 39-43, 40-44, 41-45, 42-46, 43-47, 44-48,45-49, 46-50, 47-51, 48-52, 49-53, 50-54, 51-55, 52-56, 53-57, 54-58,55-59, 56-60, 57-61, 58-62, 59-63, 60-64, 61-65, 62-66, 63-67, 64-68,65-69, 66-70, 67-71, 68-72, 69-73, 70-74, 71-75, 72-76, 73-77, 74-78,75-79, 76-80, 77-81, 78-82, 79-83, 80-84, 81-85, 82-86, 83-87, 84-88,85-89, 86-90, 87-91, 88-92, 89-93, 90-94, 91-95, 92-96, 93-97, 94-98,95-99, 96-100.

An additional set for regions 20 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:

S(20)={R _(1,20) |nε{1,2,3, . . . , 81}}

and describes the set of regions comprising nucleobases 1-20, 2-21,3-22, 4-23, 5-24, 6-25, 7-26, 8-27, 9-28, 10-29, 11-30, 12-31, 13-32,14-33, 15-34, 16-35, 17-36, 18-37, 19-38, 20-39, 21-40, 22-41, 23-42,24-43, 25-44, 26-45, 27-46, 28-47, 29-48, 30-49, 31-50, 32-51, 33-52,34-53, 35-54, 36-55, 37-56, 38-57, 39-58, 40-59, 41-60, 42-61, 43-62,44-63, 45-64, 46-65, 47-66, 48-67, 49-68, 50-69, 51-70, 52-71, 53-72,54-73, 55-74, 56-75, 57-76, 58-77, 59-78, 60-79, 61-80, 62-81, 63-82,64-83, 65-84, 66-85, 67-86, 68-87, 69-88, 70-89, 71-90, 72-91, 73-92,74-93, 75-94, 76-95, 77-96, 78-97, 79-98, 80-99, 81-100.

An additional set for regions 80 nucleobases in length, in a targetsequence 100 nucleobases in length, beginning at position 1 of thetarget nucleobase sequence, can be described using the followingexpression:

S(80)={R _(1,80) /nε{1,2,3, . . . , 21}}

and describes the set of regions comprising nucleobases 1-80, 2-81,3-82, 4-83, 5-84, 6-85, 7-86, 8-87, 9-88, 10-89, 11-90, 12-91, 13-92,14-93, 15-94, 16-95, 17-96, 18-97, 19-98, 20-99, 21-100.

Thus, in this example, A would include regions 1-5, 2-6, 3-7 . . .93-100, 1-20, 2-21, 3-22 . . . 81-100, 1-80, 2-81, 3-82 . . . 21-100.

The union of these aforementioned example sets and other sets forlengths from 10 to 19 and 21 to 79 can be described using themathematical expression

$A = {\bigcup\limits_{m}{S(m)}}$

where ∪ represents the union of the sets obtained by combining allmembers of all sets.

The mathematical expressions described herein defines all possibletarget regions in a target nucleobase sequence of any length L, wherethe region is of length m, and where m is greater than or equal to 5 andless than or equal to 80 nucleobases and, and where m is less than L,and where n is less than L−m+1.

In accordance with one embodiment of the present invention, a series ofnucleic acid duplexes comprising the chimeric oligomeric compounds ofthe present invention and their complements can be designed for aspecific target or targets. These nucleic acid duplexes are commonlyreferred to in the art as double-strand RNAs (dsRNAs) or smallinterfering RNAs (siRNAs). As described herein, such duplexes have beenshown in the art to modulate target expression and regulate translationas well as RNA processing via an antisense mechanism. Within a duplex,the ends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe duplex is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of theduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini. The antisense and sense strands of theduplex comprise from about 17 to 25 nucleotides, or from about 19 to 23nucleotides. Alternatively, the antisense and sense strands comprise 20,21 or 22 nucleotides.

For example, a duplex comprising a chimeric oligomeric compound havingthe sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 3) and having atwo-nucleobase overhang of deoxythymidine(dT) would have the followingstructure:

Overhangs can range from 1 to 6 nucleobases and these nucleobases may ormay not be complementary to the target nucleic acid. One of skill in theart will understand that the overhang may be 1, 2, 3, 4, 5 or 6nucleobases in length. In another embodiment, the duplexes may have anoverhang on only on terminus.

In another embodiment, a duplex comprising an antisense strand havingthe same sequence CGAGAGGCGGACGGGACCG (SEQ ID NO: 3) may be preparedwith blunt ends (no single stranded overhang) as shown:

The RNA duplex can be unimolecular or bimolecular; i.e., the two strandscan be part of a single molecule or may be separate molecules. Thesesequences are shown to contain thymine (T), but one of skill in the artwill appreciate that thymine (T) can generally be replaced with uracil(U) in RNA sequences.

Screening and Target Validation

In a further embodiment, “suitable target segments” may be employed in ascreen for additional oligomeric compounds that modulate the expressionof a selected protein. “Modulators” are those oligomeric compounds thatdecrease or increase the expression of a nucleic acid molecule encodinga protein and which comprise at least an 8-nucleobase portion which iscomplementary to a suitable target segment. The screening methodcomprises the steps of contacting a suitable target segment of a nucleicacid molecule encoding a protein with one or more candidate modulators,and selecting for one or more candidate modulators which decrease orincrease the expression of a nucleic acid molecule encoding a protein.Once it is shown that the candidate modulator or modulators are capableof modulating (e.g. either decreasing or increasing) the expression of anucleic acid molecule encoding a peptide, the modulator may then beemployed in further investigative studies of the function of thepeptide, or for use as a research, diagnostic, or therapeutic agent inaccordance with the present invention.

The suitable target segments of the present invention may also becombined with their respective complementary chimeric oligomericcompounds of the present invention to form stabilized double-stranded(duplexed) oligonucleotides. Such double-stranded oligonucleotidemoieties have been shown in the art to modulate target expression andregulate translation as well as RNA processing via an antisensemechanism.

The oligomeric compounds of the present invention can also be applied inthe areas of drug discovery and target validation. The present inventioncomprehends the use of the oligomeric compounds and suitable targetsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between proteins and a disease state, phenotype, orcondition. These methods include detecting or modulating a targetpeptide comprising contacting a sample, tissue, cell, or organism withthe oligomeric compounds of the present invention, measuring the nucleicacid or protein level of the target and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther oligomeric compound of the invention. These methods can also beperformed in parallel or in combination with other experiments todetermine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a particular disease, condition,or phenotype.

Kits, Research Reagents, Diagnostics, and Therapeutics

The oligomeric compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. Furthermore, antisense oligonucleotides, which are able to inhibitgene expression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the oligomeric compounds of the presentinvention, either alone or in combination with other oligomericcompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

As one nonlimiting example, expression patterns within cells or tissuestreated with one or more chimeric oligomeric compounds are compared tocontrol cells or tissues not treated with chimeric oligomeric compoundsand the patterns produced are analyzed for differential levels of geneexpression as they pertain, for example, to disease association,signaling pathway, cellular localization, expression level, size,structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds and or oligomeric compounds which affectexpression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The oligomeric compounds of the invention are useful for research anddiagnostics, because these oligomeric compounds hybridize to nucleicacids encoding proteins. The primers and probes disclosed herein areuseful in methods requiring the specific detection of nucleic acidmolecules encoding proteins and in the amplification of the nucleic acidmolecules for detection or for use in further studies. Hybridization ofthe primers and probes with a nucleic acid can be detected by meansknown in the art. Such means may include conjugation of an enzyme to theprimer or probe, radiolabelling of the primer or probe or any othersuitable detection means. Kits using such detection means for detectingthe level of selected proteins in a sample may also be prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligomeric compoundshave been employed as therapeutic moieties in the treatment of diseasestates in animals, including humans. Antisense oligonucleotide drugs,including ribozymes, have been safely and effectively administered tohumans and numerous clinical trials are presently underway. It is thusestablished that antisense oligomeric compounds can be usefultherapeutic modalities that can be configured to be useful in treatmentregimes for the treatment of cells, tissues and animals, especiallyhumans.

For therapeutics, an animal, such as a human, suspected of having adisease or disorder which can be treated by modulating the expression ofa selected protein is treated by administering chimeric oligomericcompounds in accordance with this invention. For example, in onenon-limiting embodiment, the methods comprise the step of administeringto the animal in need of treatment, a therapeutically effective amountof a protein inhibitor. The protein inhibitors of the present inventioneffectively inhibit the activity of the protein or inhibit theexpression of the protein. In one embodiment, the activity or expressionof a protein in an animal is inhibited by about 10% or more, by about20% or more, by about 30% or more, by about 40% or more, by about 50% ormore, by about 60% or more, by about 70% or more, by about 80% or more,by about 90% or more, by about 95% or more, or by about 99% or more. Forexample, the reduction of the expression of a protein may be measured inserum, adipose tissue, liver or any other body fluid, tissue or organ ofthe animal. The cells contained within the fluids, tissues or organsbeing analyzed can contain a nucleic acid molecule encoding a proteinand/or the protein itself.

The oligomeric compounds of the invention can be utilized inpharmaceutical compositions by adding an effective amount of anoligomeric compound to a suitable pharmaceutically acceptable diluent orcarrier. Use of the oligomeric compounds and methods of the inventionmay also be useful prophylactically.

Formulations

The oligomeric compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The chimeric oligomeric compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the oligomeric compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents. The term“prodrug” indicates a therapeutic agent that is prepared in an inactiveform that is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions. In particular, prodrug versions of the oligomericcompounds of the invention are prepared asSATE[(S-acetyl-2-thioethyl)phosphate]derivatives according to themethods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9,1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

Pharmaceutical Compositions and Routes of Administration

The present invention also includes pharmaceutical compositions andformulations which include the oligomeric compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Topical formulations include those in which theoligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Lipids and liposomesinclude neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Fatty acids andesters include but are not limited arachidonic acid, oleic acid,eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

In some embodiments, an oligonucleotide may be administered to a subjectvia an oral route of administration. The subjects of the presentinvention comprise animals. An animal subject may be a mammal, such as amouse, a rat, a dog, a guinea pig, a monkey, a human, a non-humanprimate, a cat or a pig. Non-human primates include monkeys andchimpanzees. A suitable animal subject may be an experimental animal,such as a mouse, a rat, a dog, a non-human primate, a cat or a pig.

In some embodiments, the subject may be a human. In certain embodiments,the subject may be a human patient as discussed in more detail herein.In certain embodiments, it may be necessary to modulate the expressionof one or more genes of the human patient. In some particularembodiments, it may be necessary to inhibit expression of one or moregenes of the human patient. In particular embodiments, it may benecessary to modulate, i e inhibit or enhance, the expression of one ormore genes in order to obtain therapeutic outcomes discussed herein.

In some embodiments, non-parenteral (e.g. oral) oligonucleotideformulations according to the present invention result in enhancedbioavailability of the oligonucleotide. In this context, the term“bioavailability” refers to a measurement of that portion of anadministered drug which reaches the circulatory system (e.g. blood,especially blood plasma) when a particular mode of administration isused to deliver the drug. Enhanced bioavailability refers to aparticular mode of administration's ability to deliver oligonucleotideto the peripheral blood plasma of a subject relative to another mode ofadministration. For example, when a non-parenteral mode ofadministration (e.g. an oral mode) is used to introduce the drug into asubject, the bioavailability for that mode of administration may becompared to a different mode of administration, e.g. an IV mode ofadministration. In some embodiments, the area under a compound's bloodplasma concentration curve (AUC₀) after non-parenteral administrationmay be divided by the area under the drug's plasma concentration curveafter intravenous (i.v.) administration (AUC_(iv)) to provide adimensionless quotient (relative bioavailability, RB) that representsfraction of compound absorbed via the non-parenteral route as comparedto the IV route. A composition's bioavailability is said to be enhancedin comparison to another composition's bioavailability when the firstcomposition's relative bioavailability (RB₁) is greater than the secondcomposition's relative bioavailability (RB₂).

In general, bioavailability correlates with therapeutic efficacy when acompound's therapeutic efficacy is related to the blood concentrationachieved, even if the drug's ultimate site of action is intracellular(van Berge-Henegouwen et al., Gastroenterol., 1977, 73, 300).Bioavailability studies have been used to determine the degree ofintestinal absorption of a drug by measuring the change in peripheralblood levels of the drug after an oral dose (DiSanto, Chapter 76 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 1451-1458).

In general, an oral composition (comprising an oligonucleotide)bioavailability is said to be “enhanced” when its relativebioavailability is greater than the bioavailability of a compositionsubstantially consisting of pure oligonucleotide, i.e. oligonucleotidein the absence of a penetration enhancer.

Organ bioavailability refers to the concentration of compound in anorgan. Organ bioavailability may be measured in test subjects by anumber of means, such as by whole-body radiography. Organbioavailability may be modified, e.g. enhanced, by one or moremodifications to the oligonucleotide, by use of one or more carriercompounds or excipients, etc. as discussed in more detail herein. Ingeneral, an increase in bioavailability will result in an increase inorgan bioavailability.

Oral oligonucleotide compositions according to the present invention maycomprise one or more “mucosal penetration enhancers,” also known as“absorption enhancers” or simply as “penetration enhancers.”Accordingly, some embodiments of the invention comprise at least oneoligonucleotide in combination with at least one penetration enhancer.In general, a penetration enhancer is a substance that facilitates thetransport of a drug across mucous membrane(s) associated with thedesired mode of administration, e.g. intestinal epithelial membranes.Accordingly it is desirable to select one or more penetration enhancersthat facilitate the uptake of an oligonucleotide, without interferingwith the activity of the oligonucleotide, and in such a manner theoligonucleotide can be introduced into the body of an animal withoutunacceptable degrees of side-effects such as toxicity, irritation orallergic response.

Embodiments of the present invention provide compositions comprising oneor more pharmaceutically acceptable penetration enhancers, and methodsof using such compositions, which result in the improved bioavailabilityof oligonucleotides administered via non-parenteral modes ofadministration. Heretofore, certain penetration enhancers have been usedto improve the bioavailability of certain drugs. See Muranishi, Crit.Rev. Ther. Drug Carrier Systems, 1990, 7, 1 and Lee et al., Crit. Rev.Ther. Drug Carrier Systems, 1991, 8, 91. It has been found that theuptake and delivery of oligonucleotides can be greatly improved evenwhen administered by non-parenteral means through the use of a number ofdifferent classes of penetration enhancers.

In some embodiments, compositions for non-parenteral administrationinclude one or more modifications to naturally-occurringoligonucleotides (i.e. full-phosphodiester deoxyribosyl orfull-phosphodiester ribosyl oligonucleotides). Such modifications mayincrease binding affinity, nuclease stability, cell or tissuepermeability, tissue distribution, or other biological orpharmacokinetic property. Modifications may be made to the base, thelinker, or the sugar, in general, as discussed in more detail hereinwith regards to oligonucleotide chemistry. In some embodiments of theinvention, compositions for administration to a subject, and inparticular oral compositions for administration to an animal (human ornon-human) subject, will comprise modified oligonucleotides having oneor more modifications for enhancing affinity, stability, tissuedistribution, or other biological property.

Suitable modified linkers include phosphorothioate linkers. In someembodiments according to the invention, the oligonucleotide has at leastone phosphorothioate linker. Phosphorothioate linkers provide nucleasestability as well as plasma protein binding characteristics to theoligonucleotide. Nuclease stability is useful for increasing the in vivolifetime of oligonucleotides, while plasma protein binding decreases therate of first pass clearance of oligonucleotide via renal excretion. Insome embodiments according to the present invention, the oligonucleotidehas at least two phosphorothioate linkers. In some embodiments, whereinthe oligonucleotide has exactly n nucleosides, the oligonucleotide hasfrom one to n−1 phosphorothioate linkages. In some embodiments, whereinthe oligonucleotide has exactly n nucleosides, the oligonucleotide hasn−1 phosphorothioate linkages. In other embodiments wherein theoligonucleotide has exactly n nucleoside, and n is even, theoligonucleotide has from 1 to n/2 phosphorothioate linkages, or, when nis odd, from 1 to (n−1)/2 phosphorothioate linkages. In someembodiments, the oligonucleotide has alternating phosphodiester (PO) andphosphorothioate (PS) linkages. In other embodiments, theoligonucleotide has at least one stretch of two or more consecutive POlinkages and at least one stretch of two or more PS linkages. In otherembodiments, the oligonucleotide has at least two stretches of POlinkages interrupted by at least on PS linkage.

In some embodiments, at least one of the nucleosides is modified on theribosyl sugar unit by a modification that imparts nuclease stability,binding affinity or some other beneficial biological property to thesugar. In some cases, the sugar modification includes a 2′-modification,e.g. the 2′-OH of the ribosyl sugar is replaced or substituted. Suitablereplacements for 2′-OH include 2′-F and 2′-arabino-F. Suitablesubstitutions for OH include 2′-O-alkyl, e.g. 2-O-methyl, and2′-O-substituted alkyl, e.g. 2′-O-methoxyethyl, 2′—NH₂,2′-O-aminopropyl, etc. In some embodiments, the oligonucleotide containsat least one 2′-modification. In some embodiments, the oligonucleotidecontains at least two 2′-modifications. In some embodiments, theoligonucleotide has at least one 2′-modification at each of the termini(i.e. the 3′- and 5′-terminal nucleosides each have the same ordifferent 2′-modifications). In some embodiments, the oligonucleotidehas at least two sequential 2′-modifications at each end of theoligonucleotide. In some embodiments, oligonucleotides further compriseat least one deoxynucleoside. In particular embodiments,oligonucleotides comprise a stretch of deoxynucleosides such that thestretch is capable of activating RNase (e.g. RNase H) cleavage of an RNAto which the oligonucleotide is capable of hybridizing. In someembodiments, a stretch of deoxynucleosides capable of activatingRNase-mediated cleavage of RNA comprises about 6 to about 16, e.g. about8 to about 16 consecutive deoxynucleosides. In further embodiments,oligonucleotides are capable of eliciting cleaveage by dsRNAse enzymeswhich act on RNA:RNA hybrids.

Oligonucleotide compositions of the present invention may be formulatedin various dosage forms such as, but not limited to, tablets, capsules,liquid syrups, soft gels, suppositories, and enemas. The term“alimentary delivery” encompasses e.g. oral, rectal, endoscopic andsublingual/buccal administration. A common requirement for these modesof administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of theoligonucleotides or mimetics thereof so administered.

Delivery of a drug via the oral mucosa, as in the case of buccal andsublingual administration, has several desirable features, including, inmany instances, a more rapid rise in plasma concentration of the drug(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).

Endoscopy may be used for drug delivery directly to an interior portionof the alimentary tract. For example, endoscopic retrogradecystopancreatography (ERCP) takes advantage of extended gastroscopy andpermits selective access to the biliary tract and the pancreatic duct(Hirahata et al., Gan To Kagaku Ryoho, 1992, 19(10 Suppl.), 1591).Pharmaceutical compositions, including liposomal formulations, can bedelivered directly into portions of the alimentary canal, such as, e.g.,the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the gastricsubmucosa (Akamo et al., Japanese J. Cancer Res., 1994, 85, 652) viaendoscopic means. Gastric lavage devices (Inoue et al., Artif. Organs,1997, 21, 28) and percutaneous endoscopic feeding devices (Pennington etal., Ailment Pharmacol. Ther., 1995, 9, 471) can also be used for directalimentary delivery of pharmaceutical compositions.

In some embodiments, oligonucleotide formulations may be administeredthrough the anus into the rectum or lower intestine. Rectalsuppositories, retention enemas or rectal catheters can be used for thispurpose and may be desired when patient compliance might otherwise bedifficult to achieve (e.g., in pediatric and geriatric applications, orwhen the patient is vomiting or unconscious). Rectal administration canresult in more prompt and higher blood levels than the oral route.(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711). Becauseabout 50% of the drug that is absorbed from the rectum will likelybypass the liver, administration by this route significantly reduces thepotential for first-pass metabolism (Benet et al., Chapter 1 In: Goodman& Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardmanet al., eds., McGraw-Hill, New York, N.Y., 1996).

Some embodiments employ various penetration enhancers in order to effecttransport of oligonucleotides and other nucleic acids across mucosal andepithelial membranes. Penetration enhancers may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92).Accordingly, some embodiments comprise oral oligonucleotide compositionscomprising at least one member of the group consisting of surfactants,fatty acids, bile salts, chelating agents, and non-chelatingsurfactants. Further embodiments comprise oral oligonucleotidecompositions comprising at least one fatty acid, e.g. capric or lauricacid, or combinations or salts thereof. Other embodiments comprisemethods of enhancing the oral bioavailability of an oligonucleotide, themethod comprising co-administering the oligonucleotide and at least onepenetration enhancer.

Other excipients that may be added to oral oligonucleotide compositionsinclude surfactants (or “surface-active agents”). These are chemicalentities which, when dissolved in an aqueous solution, reduce thesurface tension of the solution or the interfacial tension between theaqueous solution and another liquid, with the result that absorption ofoligonucleotides through the alimentary mucosa and other epithelialmembranes is enhanced. In addition to bile salts and fatty acids,surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorohemical emulsions, such as FC-43 (Takahashi et al., J.Pharm. Phamacol., 1988, 40, 252).

Fatty acids and their derivatives which act as penetration enhancers andmay be used in compositions of the present invention include, forexample, oleic acid, lauric acid, capric acid (n-decanoic acid),myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol),dilaurin, caprylic acid, arachidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines and mono-and di-glycerides thereof and/or physiologically acceptable saltsthereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate,linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

In some embodiments, oligonucleotide compositions for oral deliverycomprise at least two discrete phases, which phases may compriseparticles, capsules, gel-capsules, microspheres, etc. Each phase maycontain one or more oligonucleotides, penetration enhancers,surfactants, bioadhesives, effervescent agents, or other adjuvant,excipient or diluent. In some embodiments, one phase comprises at leastone oligonucleotide and at lease one penetration enhancer. In someembodiments, a first phase comprises at least one oligonucleotide and atleast one penetration enhancer, while a second phase comprises at leastone penetration enhancer. In some embodiments, a first phase comprisesat least one oligonucleotide and at least one penetration enhancer,while a second phase comprises at least one penetration enhancer andsubstantially no oligonucleotide. In some embodiments, at least onephase is compounded with at least one degradation retardant, such as acoating or a matrix, which delays release of the contents of that phase.In some embodiments, at least one phase In some embodiments, a firstphase comprises at least one oligonucleotide, at least one penetrationenhancer, while a second phase comprises at least one penetrationenhancer and a release-retardant. In particular embodiments, an oraloligonucleotide composition comprises a first phase comprising particlescontaining an oligonucleotide and a penetration enhancer, and a secondphase comprising particles coated with a release-retarding agent andcontaining penetration enhancer.

A variety of bile salts also function as penetration enhancers tofacilitate the uptake and bioavailability of drugs. The physiologicalroles of bile include the facilitation of dispersion and absorption oflipids and fat-soluble vitamins (Brunton, Chapter 38 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., eds., McGraw-Hill, New York, N.Y., 1996, pages 934-935). Variousnatural bile salts, and their synthetic derivatives, act as penetrationenhancers. Thus, the term “bile salt” includes any of the naturallyoccurring components of bile as well as any of their syntheticderivatives. The bile salts of the invention include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(CDCA, sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579).

In some embodiments, penetration enhancers of the present invention aremixtures of penetration enhancing compounds. One such penetrationmixture is UDCA (and/or CDCA) with capric and/or lauric acids or saltsthereof e.g. sodium. Such mixtures are useful for enhancing the deliveryof biologically active substances across mucosal membranes, inparticular intestinal mucosa. Other penetration enhancer mixturescomprise about 5-95% of bile acid or salt(s) UDCA and/or CDCA with 5-95%capric and/or lauric acid. Particular penetration enhancers are mixturesof the sodium salts of UDCA, capric acid and lauric acid in a ratio ofabout 1:2:2 respectively. Anther such penetration enhancer is a mixtureof capric and lauric acid (or salts thereof) in a 0.01:1 to 1:0.01 ratio(mole basis). In particular embodiments capric acid and lauric acid arepresent in molar ratios of e.g. about 0.1:1 to about 1:0.1, inparticular about 0.5:1 to about 1:0.5.

Other excipients include chelating agents, i.e. compounds that removemetallic ions from solution by forming complexes therewith, with theresult that absorption of oligonucleotides through the alimentary andother mucosa is enhanced. With regards to their use as penetrationenhancers in compositions containing DNA-like oligonucleotides in thepresent invention, chelating agents have the added advantage of alsoserving as DNase inhibitors, as most characterized DNA nucleases requirea divalent metal ion for catalysis and are thus inhibited by chelatingagents (Jarrett, J. Chromatogr., 1993, 618, 315). Chelating agents ofthe invention include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. ControlRel., 1990, 14, 43).

As used herein, non-chelating non-surfactant penetration enhancers maybe defined as compounds that demonstrate insignificant activity aschelating agents or as surfactants but that nonetheless enhanceabsorption of oligonucleotides through the alimentary and other mucosalmembranes (Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1). This class of penetration enhancers includes, butis not limited to, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical, therapeutic and other compositionsof the present invention. For example, cationic lipids, such aslipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerolderivatives, and polycationic molecules, such as polylysine (Lollo etal., PCT Application WO 97/30731), can be used.

A “pharmaceutical carrier” or “excipient” may be a pharmaceuticallyacceptable solvent, suspending agent or any other pharmacologicallyinert vehicle for delivering one or more nucleic acids to an animal. Theexcipient may be liquid or solid and is selected, with the plannedmanner of administration in mind, so as to provide for the desired bulk,consistency, etc., when combined with a an oligonucleotide and the othercomponents of a given pharmaceutical composition. Typical pharmaceuticalcarriers include, but are not limited to, binding agents (e.g.,pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose, etc.); fillers (e.g., lactose and other sugars,microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, EXPLOTAB);and wetting agents (e.g., sodium lauryl sulphate, etc.).

Oral oligonucleotide compositions may additionally contain other adjunctcomponents conventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions maycontain additional, compatible, pharmaceutically-active materials suchas, for example, antipruritics, astringents, local anesthetics oranti-inflammatory agents, or may contain additional materials useful inphysically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 um indiameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides from thegastrointestinal tract, as well as improve the local cellular uptake ofoligonucleotides within the gastrointestinal tract, vagina, buccalcavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used in the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes and are useful for the delivery ofDNA, RNA or any nucleic acid-based construct. Cationic liposomes arepositively charged liposomes which interact with negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

WO 96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include an antisense RNA. U.S. Pat. No.5,665,710 to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising antisense oligonucleotides targeted to the rafgene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides. The use of surfactants in drugproducts, formulations and in emulsions has been reviewed (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Pulsatile Delivery

The compounds of the present invention may also be administered bypulsatile delivery. “Pulsatile delivery” refers to a pharmaceuticalformulation that delivers a first pulse of drug (e.g. an antisensecompound) combined with a penetration enhancer and a second pulse ofpenetration enhancer to promote absorption of drug which is not absorbedupon release with the first pulse of penetration enhancer.

One embodiment of the present invention is a delayed release oralformulation for enhanced intestinal drug absorption, comprising:

(a) a first population of carrier particles comprising said drug and apenetration enhancer, wherein said drug and said penetration enhancerare released at a first location in the intestine; and

(b) a second population of carrier particles comprising a penetrationenhancer and a delayed release coating or matrix, wherein thepenetration enhancer is released at a second location in the intestinedownstream from the first location, whereby absorption of the drug isenhanced when the drug reaches the second location.

Alternatively, the penetration enhancer in (a) and (b) is different.

This enhancement is obtained by encapsulating at least two populationsof carrier particles. The first population of carrier particlescomprises a biologically active substance and a penetration enhancer,and the second (and optionally additional) population of carrierparticles comprises a penetration enhancer and a delayed release coatingor matrix.

A “first pass effect” that applies to orally administered drugs isdegradation due to the action of gastric acid and various digestiveenzymes. One means of ameliorating first pass clearance effects is toincrease the dose of administered drug, thereby compensating forproportion of drug lost to first pass clearance. Although this may bereadily achieved with i.v. administration by, for example, simplyproviding more of the drug to an animal, other factors influence thebioavailability of drugs administered via non-parenteral means. Forexample, a drug may be enzymatically or chemically degraded in thealimentary canal or blood stream and/or may be impermeable orsemipermeable to various mucosal membranes.

It is also contemplated that these pharmaceutical compositions arecapable of enhancing absorption of biologically active substances whenadministered via the rectal, vaginal, nasal or pulmonary routes. It isalso contemplated that release of the biologically active substance canbe achieved in any part of the gastrointestinal tract.

Liquid pharmaceutical compositions of oligonucleotide can be prepared bycombining the oligonucleotide with a suitable vehicle, for examplesterile pyrogen free water, or saline solution. Other therapeuticcompounds may optionally be included.

The present invention also contemplates the use of solid particulatecompositions. Such compositions comprise particles of oligonucleotidethat are of respirable size. Such particles can be prepared by, forexample, grinding dry oligonucleotide by conventional means, foreexample with a mortar and pestle, and then passing the resulting powdercomposition through a 400 mesh screen to segregate large particles andagglomerates. A solid particulate composition comprised of an activeoligonucleotide can optionally contain a dispersant which serves tofacilitate the formation of an aerosol, for example lactose.

In accordance with the present invention, oligonucleotide compositionscan be aerosolized. Aerosolization of liquid particles can be producedby any suitable means, such as with a nebulizer. See, for example, U.S.Pat. No. 4,501,729. Nebulizers are commercially available devices whichtransform solutions or suspensions into a therapeutic aerosol misteither by means of acceleration of a compressed gas, typically air oroxygen, through a narrow venturi orifice or by means of ultrasonicagitation. Suitable nebulizers include those sold by Blairex® under thename PARI LC PLUS, PARI DURA-NEB 2000, PARI-BABY Size, PARI PRONEBCompressor with LC PLUS, PARI WALKHALER Compressor/Nebulizer System,PARI LC PLUS Reusable Nebulizer, and PARI LC Jet+®Nebulizer.

Formulations for use in nebulizers may consist of an oligonucleotide ina liquid, such as sterile, pyragen free water, or saline solution,wherein the oligonucleotide comprises up to about 40% w/w of theformulation. The oligonucleotide can comprise less than 20% w/w. Ifdesired, further additives such as preservatives (for example, methylhydroxybenzoate) antioxidants, and flavoring agents can be added to thecomposition.

Solid particles comprising an oligonucleotide can also be aerosolizedusing any solid particulate medicament aerosol generator known in theart. Such aerosol generators produce respirable particles, as describedabove, and further produce reproducible metered dose per unit volume ofaerosol. Suitable solid particulate aerosol generators includeinsufflators and metered dose inhalers. Metered dose inhalers are usedin the art and are useful in the present invention.

Liquid or solid aerosols are produced at a rate of from about 10 to 150liters per minute, from about 30 to 150 liters per minute, or from about60 to 150 liters per minute.

Enhanced bioavailability of biologically active substances is alsoachieved via the oral administration of the compositions and methods ofthe present invention.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Two or more antisensecompounds may be used together or sequentially.

Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 μgto 100 g per kg of body weight, from 0.1 μg to 10 g per kg of bodyweight, from 1.0 μg to 1 g per kg of body weight, from 10.0 μg to 100 mgper kg of body weight, from 100 μg to 10 mg per kg of body weight, orfrom 1 mg to 5 mg per kg of body weight, and may be given once or moredaily, weekly, monthly or yearly, or even once every 2 to 20 years.Persons of ordinary skill in the art can easily estimate repetitionrates for dosing based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state, wherein the oligonucleotideis administered in maintenance doses, ranging from 0.01 ug to 100 g perkg of body weight, once or more daily, to once every 20 years.

The effects of treatments with therapeutic compositions can be assessedfollowing collection of tissues or fluids from a patient or subjectreceiving said treatments. It is known in the art that a biopsy samplecan be procured from certain tissues without resulting in detrimentaleffects to a patient or subject. In certain embodiments, a tissue andits constituent cells comprise, but are not limited to, blood (e.g.,hematopoietic cells, such as human hematopoietic progenitor cells, humanhematopoietic stem cells, CD34+ cells CD4+ cells), lymphocytes and otherblood lineage cells, bone marrow, breast, cervix, colon, esophagus,lymph node, muscle, peripheral blood, oral mucosa and skin. In otherembodiments, a fluid and its constituent cells comprise, but are notlimited to, blood, urine, semen, synovial fluid, lymphatic fluid andcerebro-spinal fluid. Tissues or fluids procured from patients can beevaluated for expression levels of the target mRNA or protein.Additionally, the mRNA or protein expression levels of other genes knownor suspected to be associated with the specific disease state, conditionor phenotype can be assessed. mRNA levels can be measured or evaluatedby real-time PCR, Northern blot, in situ hybridization or DNA arrayanalysis. Protein levels can be measured or evaluated by ELISA,immunoblotting, quantitative protein assays, protein activity assays(for example, caspase activity assays) immunohistochemistry orimmunocytochemistry. Furthermore, the effects of treatment can beassessed by measuring biomarkers associated with the disease orcondition in the aforementioned tissues and fluids, collected from apatient or subject receiving treatment, by routine clinical methodsknown in the art. These biomarkers include but are not limited to:glucose, cholesterol, lipoproteins, triglycerides, free fatty acids andother markers of glucose and lipid metabolism; lipoprotein(a) particleand apolipoprotein B-100; liver transaminases, bilirubin, albumin, bloodurea nitrogen, creatine and other markers of kidney and liver function;interleukins, tumor necrosis factors, intracellular adhesion molecules,C-reactive protein and other markers of inflammation; testosterone,estrogen and other hormones; tumor markers; vitamins, minerals andelectrolytes.

The present invention also provides methods of reducing target RNAlevels in an animal comprising contacting the animal with a gap-disabledcompound comprising a gap-disabled motif listed in Table 13 or Table 26and wherein the gap-disabled compound comprises a nucleobase sequencesubstantially complementary to a portion of the target RNA. Thesemethods may also comprise identifying an animal in need of reducingtarget RNA levels.

The present invention also provides methods of lowering cholesterol ortriglycerides in an animal comprising contacting the animal with agap-disabled compound comprising the gap-disabled motif3-2-1-2-1-2-1-2-1-2-3. These methods may also comprise identifying ananimal in need of lowering cholesterol or triglycerides.

The present invention also provides methods of lowering plasma leptin,glucose, or plasma insulin in an animal comprising contacting the animalwith a gap-disabled compound having the gap-disabled motif3-2-1-2-1-2-1-2-1-2-3. These methods may also comprise identifying ananimal in need of lowering plasma leptin, glucose, or plasma insulin.

The present invention also provides methods of lowering body weight, fatdepot weight or food intake in an animal comprising contacting theanimal with a gap-disabled compound comprising the gap-disabled motif3-2-1-2-1-2-1-2-1-2-3. These methods may also comprise identifying ananimal in need of lowering body weight, fat depot weight or food intake.

The present invention also provides methods of reducing serumcholesterol, triglycerides or body weight in an obese animal comprisingcontacting the animal with a gap-disabled compound comprising thegap-disabled motif of 3-2-1-2-1-2-1-2-1-2-3. These methods may alsocomprise identifying an obese animal in need of reducing serumcholesterol, triglycerides or body weight.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner. Throughout theseexamples, molecular cloning reactions, and other standard recombinantDNA techniques, were carried out according to methods described inManiatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., ColdSpring Harbor Press (1989), using commercially available reagents,except where otherwise noted.

EXAMPLES Examples 1-17 Scheme I, FIGS. 1-3 Preparation of1-(8-hydroxy-5-hydroxymethyl-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]oct-7-yl)-1H-pyrimidine-2,4-dione(1)

Example 11-(3-hydroxy-5,5,7,7-tetraisopropyl-tetrahydro-1,4,6,8-tetraoxa-5,7-disila-cyclopentacycloocten-2-yl)-1H-pyrimidine-2,4-dione(4)

The 3′,5′-protected nucleoside is prepared as illustrated in Karpeisky,A., et. al., Tetrahedron Lett. 1998, 39, 1131-1134. To a solution ofarabinouridine (3, 1.0 eq., 0° C.) in anhydrous pyridine is added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.1 eq.). The resultingsolution is warmed to room temperature and stirred for two hours. Thereaction mixture is subsequently quenched with methanol, concentrated toan oil, dissolved in dichloromethane, washed with aqueous NaHCO₃ andsaturated brine, dried over anhydrous Na₂SO₄, filtered, and evaporated.Purification by silica gel chromatography will yield Compound 4.

For the preparation of the corresponding cytidine and adenosine analogs,N⁴-benzoyl arabinocytidine and N⁶-benzoyl arabinoadenosine are used,respectively, both of which are prepared from the unprotectedarabinonucleoside using the transient protection strategy as illustratedin Ti, et al., J. Am. Chem. Soc. 1982, 104, 1316-1319. Alternatively,the cytidine analog can also be prepared by conversion of the uridineanalog as illustrated in Lin, et al., J. Med. Chem. 1983, 26, 1691.

Example 2 Acetic acid2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-5,5,7,7-tetraisopropyl-tetrahydro-1,4,6,8-tetraoxa-5,7-disila-cyclopentacycloocten-3-ylester (5)

Compound 4 is O-Acetylated using well known literature procedures(Protective Groups in Organic Synthesis, 3^(rd) edition, 1999, pp.150-160 and references cited therein and in Greene, T. W. and Wuts, P.G. M., eds, Wiley-Interscience, New York.) Acetic anhydride (2 to 2.5eq.) and triethylamine (4 eq.) is added to a solution of 4 (1 eq.) andN,N-dimethylaminopyridine (0.1 eq.) in anhydrous pyridine. Afterstirring at room temperature for 1 hour the mixture is treated withmethanol to quench excess acetic anhydride and evaporated. The residueis redissolved in ethyl acetate, washed extensively with aqueous NaHCO₃,dried over anhydrous Na₂SO₄, filtered, and evaporated. The compound isused without further purification.

Example 3 Acetic acid2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-hydroxy-5-hydroxymethyl-tetrahydro-furan-3-ylester (6)

The Tips protecting group is removed from Compound 5 as illustrated inthe literature (Protective Groups in Organic Synthesis, 3^(rd) edition,1999, pp. 239 and references therein, Greene, T. W. and Wuts, P. G. M.,eds, Wiley-Interscience, New York). To a solution of 5 (1 eq.) inanhydrous dichloromethane is added triethylamine (2 eq.) andtriethylamine trihydrofluoride (2 eq.). The reaction mixture ismonitored by thin layer chromatography until complete at which point thereaction mixture is diluted with additional dichloromethane, washed withaqueous NaHCO₃, dried over anhydrous Na₂SO₄, and evaporated. Theresulting Compound 6 is optionally purified by silica gelchromatography.

Example 4 Acetic acid5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-4-hydroxy-tetrahydro-furan-3-ylester (7)

Dimethoxytritylation of Compound 6 is performed using known literatureprocedures. Formation of the primary 4,4′-dimethoxytrityl ether shouldbe achieved using standard conditions (Nucleic Acids in Chemistry andBiology, 1992, pp. 108-110, Blackburn, Michael G., and Gait, Michael J.,eds, IRL Press, New York.) Generally, a solution of 6 (1 eq.) andN,N-dimethylaminopyridine (0.1 eq.) in anhydrous pyridine is treatedwith 4,4′-dimethoxytrityl chloride (DMTCl, 1.2 eq.) and triethylamine (4eq.). After several hours at room temperature, excess4,4′-dimethoxytrityl chloride is quenched with the addition of methanoland the mixture is evaporated. The mixture is dissolved indichloromethane and washed extensively with aqueous NaHCO₃ and driedover anhydrous Na₂SO₄. Purification by silica gel chromatography willyield Compound 7.

Example 5 Acetic acid5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-(tert-butyl-diphenyl-silanyloxy)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-ylester (8)

The preparation of tert-butyldiphenylsilyl ethers is a common, routineprocedure (Protective Groups in Organic Synthesis, 3^(rd) edition, 1999,pp. 141-144 and references therein, Greene, T. W. and Wuts, P. G. M.,eds, Wiley-Interscience, New York). In general, a solution of one eq. of7 and imidazole (3.5 eq.) in anhydrous N,N-dimethylformamide (DMF) istreated with tert-butyldiphenylsilyl chloride (1.2 eq.). After stirringat room temperature for several hours, the reaction mixture is pouredinto ethyl acetate and washed extensively with water and saturated brinesolution. The resulting organic solution is dried over anhydrous sodiumsulfate, filtered, evaporated, and purified by silica gel chromatographyto give Compound 8.

Example 6 Acetic acid4-(tert-butyl-diphenyl-silanyloxy)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-5-hydroxymethyl-tetrahydro-furan-3-ylester (9)

The 5′-O-DMT group is removed as per known literature procedures4,4′-dimethoxytrityl ethers are commonly removed under acidic conditions(Oligonucleotides and analogues, A Practical Approach, Eckstein, F., ed,IRL Press, New York.) Generally, Compound 8 (1 eq.) is dissolved in 80%aqueous acetic acid. After several hours, the mixture is evaporated,dissolved in ethyl acetate and washed with a sodium bicarbonatesolution. Purification by silica gel chromatography will give compound9.

Example 7 Acetic acid4-(tert-butyl-diphenyl-silanyloxy)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-5-formyl-tetrahydro-furan-3-ylester (10)

To a mixture of trichloroacetic anhydride (1.5 eq.) anddimethylsulfoxide (2.0 eq.) in dichloromethane at −78° C. is added asolution of Compound 9 in dichloromethane. After 30 minutes,triethylamine (4.5 eq.) is added. Subsequently, the mixture is pouredinto ethyl acetate, washed with water and brine, dried over anhydroussodium sulfate, and evaporated to dryness. The resulting material iscarried into the next step without further purification. This procedurehas been used to prepare the related 4′-C-∀-formyl nucleosides (Nomura,M., et. al., J. Med. Chem. 1999, 42, 2901-2908).

Example 81-[4-(tert-butyl-diphenyl-silanyloxy)-3-hydroxy-5,5-bis-hydroxymethyl-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione(11)

Hydroxymethylation of the 5′-aldehyde is performed as per the method ofCannizzaro which is well documented in the literature (Jones, G. H., et.al., J. Org. Chem. 1979, 44, 1309-1317). These condisions are expectedto additionally remove the 2′-O-acetyl group. Generally, Briefly,formaldehyde (2.0 eq., 37% aq.) and NaOH (1.2 eq., 2 M) is added to asolution of Compound 10 in 1,4-dioxane. After stirring at roomtemperature for several hours, this mixture is neutralized with aceticacid, evaporated to dryness, suspended in methanol, and evaporated ontosilica gel. The resulting mixture is added to the top of a silica gelcolumn and eluted using an appropriate solvent system to give Compound11.

Example 91-[5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-(tert-butyl-diphenyl-silanyloxy)-3-hydroxy-5-hydroxymethyl-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione(12)

Preferential protection with DMT at the ∀-hydroxymethyl position isperformed following a published literature procedure (Nomura, M., et.al., J. Med. Chem. 1999, 42, 2901-2908). Generally, a solution ofCompound II (1 eq.) in anhydrous pyridine is treated with DMTC1 (1.3eq.), then stirred at room temperature for several hours. Subsequently,the mixture is poured into ethyl acetate, washed with water, dried overanhydrous Na₂SO₄, filtered, and evaporated. Purification by silica gelchromatography will yield Compound 12.

Example 101-[5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-3-hydroxy-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione(13)

The 5′-hydroxyl position is selectively protected withtert-butyldiphenylsilyl following published literature procedures(Protective Groups in Organic Synthesis, 3^(rd) edition, 1999, pp.141-144 and references therein, Greene, T. W. and Wuts, P. G. M., eds,Wiley-Interscience, New York). Generally, a solution of Compound 12 (1eq.) and N,N-dimethylaminopyridine (0.2 eq.) in anhydrousdichloromethane is treated with tert-butyldiphenylsilyl chloride (1.2eq.) and triethylamine (4 eq.). After several hours at room temperature,the reaction is quenched with methanol, poured into ethyl acetate,washed with saturated NaHCO₃, saturated brine, dried over anhydrousNa₂SO₄, filtered, and evaporated. Purification by silica gelchromatography will yield Compound 13.

Example 11 Acetic acid5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-ylester (14)

Compound 14 is prepared as per the procedure illustrated in Example 2above.

Example 12 Acetic acid4-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-5-hydroxymethyl-tetrahydro-furan-3-ylester (15)

Compound 15 is prepared as per the procedure illustrated in Example 9above.

Example 13 Acetic acid4-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-5-(1,3-dioxo-1,3-dihydro-isoindol-2-yloxymethyl)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-tetrahydro-furan-3-ylester (16)

The use of the Mitsunobu procedure to generate the 5′-O-phthalimidonucleosides starting with the 5′-unprotected nucleosides has beenreported previously (Perbost, M., et. al., J. Org. Chem. 1995, 60,5150-5156). Generally, a mixture of Compound 15 (1 eq.),triphenylphosphine (1.15 eq.), and N-hydroxyphthalimide (PhthNOH, 1.15eq.) in anhydrous 1,4-dioxane is treated with diethyl azodicarboxylate(DEAD, 1.15 eq.). The reaction is stirred at room temperature forseveral hours until complete by thin layer chromatography. The resultingmixture is evaporated, suspended in ethyl acetate, washed with bothsaturated NaHCO₃ and saturated brine, dried over anhydrous Na₂SO₄,filtered and evaporated. Purification by silica gel chromatography willyield Compound 16.

Example 141-[4-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-3-hydroxy-5-methyleneaminooxymethyl-tetrahydro-furan-2-yl]-1H-pyrimidine-2,4-dione(17)

This transformation is performed smoothly in high yield using publishedprocedures (Bhat, B., et. al., J. Org. Chem. 1996, 61, 8186-8199).Generally, a portion of Compound 16 is dissolved in dichloromethane andcooled to −10° C. To this solution is added methylhydrazine (2.5 eq.).After 1-2 hours of stirring at 0° C., the mixture is diluted withdichloromethane, washed with water and brine, dried with anhydrousNa₂SO₄, filtered, and evaporated. The resulting residue is immediatelyredissolved in a 1:1 mixture of ethyl acetate:methanol, and treated with20% (w/w) aqueous formaldehyde (1.1 eq.). After an hour at roomtemperature, the mixture is concentrated then purified by silica gelchromatography to give Compound 17.

Example 15 Methanesulfonic acid4-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-2-(2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-5-methyleneaminooxymethyl-tetrahydro-furan-3-ylester (18)

The mesylation of hydroxyl groups proceeds readily under theseconditions (Protective Groups in Organic Synthesis, 3^(rd) edition,1999, pp. 150-160 and references cited therein). Briefly, to a solutionof Compound 17 in a 1:1 mixture of anhydrous dichloromethane andanhydrous pyridine is added methanesulfonyl chloride (1.2 eq.). Afterstirring at room temperature for several hours, this mixture is quenchedwith methanol, concentrated, diluted with dichloromethane, washed withaqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄, filtered andevaporated. Purification by silica gel chromatography will yieldCompound 18.

Example 161-[8-(tert-butyl-diphenyl-silanyloxy)-5-(tert-butyl-diphenyl-silanyloxymethyl)-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]oct-7-yl]-1H-pyrimidine-2,4-dione(19)

The reduction of the formaldoxime moiety is performed as per knownliterature procedures. Generally, a solution of Compound 18 in methanolis treated with sodium cyanoborohydride (1.5 eq.). This treatment willresult in quantitative reduction of the formaldoxime moiety to yield the4′-C-(aminooxymethyl) arabinonucleoside. The proximity of the methylatedelectron-rich amine to the activated 2′-O-mesylate will result in thespontaneous ring closing of this intermediate to yield bicyclic Compound19. The reaction is monitored by thin layer chromatography untilcompletion. The mixture is then poured into ethyl acetate, washedextensively with aqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄,filtered and evaporated. Purification by silica gel chromatography willyield Compound 19.

Example 171-(8-hydroxy-5-hydroxymethyl-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]oct-7-yl)-1H-pyrimidine-2,4-dione(1)

The tert-butyldiphenylsilyl ether protecting groups are readily cleavedby treatment with tetrabutylammonium fluoride (Protective Groups inOrganic Synthesis, 3^(rd) edition, 1999, pp. 141-144 and referencestherein, Greene, T. W. and Wuts, P. G. M., eds, Wiley-Interscience, NewYork). Briefly, a solution of Compound 19 in a minimal amount oftetrahydrofuran (THF) is treated with a 1 M solution oftetrabutylammonium fluoride (TBAF, 5-10 eq.) in THF. After several hoursat room temperature, this mixture is evaporated onto silica gel andsubjected to silica gel chromatography to give Compound 1.

Alternate Synthetic Route to Compound I, Synthesis of Guanosine AnalogExamples 18-25 Scheme II, FIGS. 4-7 Example 184-benzyloxy-5-benzyloxymethyl-5-hydroxymethyl-2-methoxy-tetrahydro-furan-3-ol(21)

The preparation of the protected 4′-C-hydroxymethylribofuranose,Compound 20, follows published literature procedures (Koshkin, A. A.,et. al., Tetrahedron 1998, 54, 3607-3630). Compound 20 (1 eq.) isdissolved in anhydrous methanol and hydrogen chloride in an anhydroussolvent (either methanol or 1,4-dioxane) is added to give a finalconcentration of 5% (w/v). After stirring at room temperature forseveral hours, the mixture is concentrated to an oil, dried undervacuum, and used in the next step without further purification.

Example 192-(3-benzyloxy-2-benzyloxymethyl-4-hydroxy-5-methoxy-tetrahydro-furan-2-ylmethoxy)-isoindole-1,3-dione(22)

The O-phthalimido compound is prepared following the reference cited andthe procedures illustrated in Example 13 above. The reaction can beadjusted to preferentially react at the primary hydroxyl e.g. the4′-C-hydroxymethyl group (Bhat, B., et. al., J. Org. Chem. 1996, 61,8186-8199). Generally, a solution of 21 (1 eq.), N-hydroxyphthalimide(1.1 eq.), and triphenylphosphine (1.1 eq.) in anhydrous tetrahydrofuranis treated with diethyl azodicarboxylate (1.1 eq.). After several hoursat room temperature, the mixture is concentrated and subjected to silicagel chromatography to give Compound 22.

Example 20 FormaldehydeO-(3-benzyloxy-2-benzyloxymethyl-4-hydroxy-5-methoxy-tetrahydro-furan-2-ylmethyl)-oxime(23)

Compound 23 is prepared as per the procedure illustrated in Example 14above.

Example 21 Methanesulfonic acid4-benzyloxy-5-benzyloxymethyl-2-methoxy-5-methylene-aminooxymethyl-tetrahydro-furan-3-ylester (24)

Mesylation is achieved with inversion of configuration using Mitsunobuconditions (Anderson, N. G., et. al., J. Org. Chem. 1996, 60, 7955).Generally, a mixture of Compound 23 (1 eq.), triphenylphosphine (1.2eq.) and methanesulfonic acid (1.2 eq.) in anhydrous 1,4-dioxane istreated with diethyl azodicarboxylate (1.2 eq.). After stirring at roomtemperature for several hours, the resulting mixture is concentrated andsubjected to silica gel chromatography to give Compound 24.

Example 228-benzyloxy-5-benzyloxymethyl-7-methoxy-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]octane(25)

Compound 25 is prepared as per the procedure illustrated in Example 16above.

Example 23 Acetic acid8-benzyloxy-5-benzyloxymethyl-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]oct-7-ylester (26)

Compound 25 is dissolved in 80% (v/v) aqueous acetic acid. After 1-2hours at room temperature, the solution is concentrated, then dissolvedin dichloromethane and washed with saturated aqueous NaHCO₃ and brine.The organic portion is subsequently dried over anhydrous Na₂SO₄,filtered, and concentrated. The resulting mixture is coevaporated fromanhydrous pyridine, then dissolved in anhydrous pyridine and treatedwith acetic anhydride (2 eq.). The solution is stirred overnight,quenched with methanol, dissolved in ethyl acetate and washedextensively with saturated NaHCO₃. The organic portion is then dried(Na₂SO₄), filtered and evaporated without further purification.

Example 241-(8-benzyloxy-5-benzyloxymethyl-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]oct-7-yl)-1H-pyrimidine-2,4-dione(27)

Compound 26 is converted to one of several N-glycosides (nucleosides)using published chemistry procedures including either Vorbrüggenchemistry or one of several other methods (Chemistry of Nucleosides andNucleotides, Volume 1, 1988, edited by Leroy B. Townsend, Plenum Press,New York). To prepare the uradinyl analog, a mixture of Compound 26 (1eq.) and uracil (1.3 eq.) is suspended in anhydrous acetonitrile. To thesuspension is added N,O-bis-(trimethylsilyl)-acetamide (BSA, 4 eq.). Thesuspension is heated to 70° C. for 1 hour, then cooled to 0° C. andtreated with trimethylsilyl-trifluoromethanesulfonate (TMSOTf, 1.6 eq.).The resulting solution is heated at 55° C. until the reaction appearscomplete by TLC. The reaction mixture is poured into ethyl acetate andwashed extensively with saturated NaHCO₃, dried over anhydrous Na₂SO₄,filtered, evaporated, and purified by silica gel chromatography to giveCompound 24.

In order to use the above preparation with nucleobases with reactivefunctional groups the reactive functional groups are protected prior touse. For example such protected nucleobases include naturally occurringnucleobases such as N⁴-benzoyl cytosine, N⁶-benzoyl adenine andN²-isobutyryl guanine

Example 251-(8-hydroxy-5-hydroxymethyl-2-methyl-3,6-dioxa-2-aza-bicyclo[3.2.1]oct-7-yl)-1H-pyrimidine-2,4-dione(1)

To give the desired product, Compound I the benzyl ethers protectinggroups are removed following published literature procedures (Koshkin,A. A., et. al., Tetrahedron 1998, 54, 3607-3630).

Generally, the bis-O-benzylated bicyclic Compound 27 is dissolved inmethanol. To this solution is added 20% Pd(OH)₂/C, and the resultingsuspension is maintained under an atmosphere of H₂ at 1-2 atm pressure.This mixture is stirred at room temperature for several hours untilcomplete by TLC, at which point the Pd(OH)₂/C is removed by filtration,and the filtrate is concentrated and purified by silica gelchromatography, if necessary, to give Compound 1.

Example 26 2′-O-tert-butyldimethylsilyl-3′-C-styryluridine (33)

Compound 28 is treated with DMTCl, in pyridine in presence of DMAP toget 5′-DMT derivative, Compound 29. Compound 29 is treated with TBDMSClin pyridine to which yields both the 2′ and the 3′-silyl derivative. The3′-TBDMS derivative is isolated by silica gel flash columnchromatography and further heated with phenyl chlorothionoformate andN-chlorosuccinimide in a solution of pyridine in benzene 60° C. to giveCompound 31. Compound 31 is treated with β-tributylstannylstyrene andAIBN in benzene give Compound 32. Compound 32 is detritylated withdichloroacetic acid in dichloromethane give compound 33.

Example 271-[(1R,3R,8S)-8-[(2-cyanoethyl)bis(1-methylethyl)phosphoramidite)-3-[(4,4′-dimethoxytrityloxy)methyl]-5-methyl-2-oxo-5-azabicyclo[2.3.1]octane-5-methyl-2,4-(1H,3H)-pyrimidinedione(40)

Compound 33 is treated with oxalyl chloride in DMSO in the presence ofethyl diisopropylamine to give the 5′-aldehyde which is then subjectedto a tandem aldol condensation and Cannizzaro reaction using aqueousformaldehyde and 1 M NaOH in 1,4-dioxane to yield the diol, Compound 34.Selective silylation with TBDMSCl in pyridine and isolation of therequired isomer will give Compound 35. Compound 35 is treated withmethanesulfonyl chloride in pyridine to give the methane sulfonylderivative which is treated with methanolic ammonia to give compound 36.The double bond of Compound 36 is oxidatively cleaved by oxymylation gogive the diol and then by cleavage of the diol with sodium periodate togive the aldehyde, Compound 37. The amino and aldehyde groups inCompound 37 are cross coupled under reductive condition followed bymethylation of the amino group with formaldehyde in the presence ofsodium borohydride will give the Compound 38. Treatment of Compound 38with triethylamine trihydrofluoride and triethylamine in THF will giveCompound 39. The primary alcohol of Compound 39 is selectively titylatedwith DMTCl in pyridine followed by phosphytilation at 8-position to giveCompound 40.

Example 281-[(1R,3R,8S)-8-[(2-cyanoethyl)bis(1-methylethyl)phosphoramidite)-3-[(4,4′-dimethoxytrityloxy)methyl]-5-methyl-2-oxo-5-azabicyclo[3.2.1]octan-4-one-5-methyl-2,4-(1H,3H)-pyrimidinedione(47)

Compound 35 is benzylated with benzyl bromide in DMF and sodium hydrideto give Compound 41. Oxidative cleavage of Compound 41 will give analdehyde at the 2′-position which is reduced to the correspondingalcohol using sodium borohydride in methanol to give Compound 42.Compound 42 is converted into the 3′-C-aminomethyl derivative, Compound43 by in situ generation of the methane sulfonyl derivative andtreatment with ammonia. The amino group in Compound 43 is protected withan Fmoc protecting group using Fmoc-Cl and sodium bicarbonate in aqueousdioxane to give Compound 44. Deprotection of the benzyl group isachieved with BCl₃ in dichloromethane at −78° C. followed by oxidationof the alcohol with pyridinium dichromate in DMF give the correspondingcarboxylic acid. The deprotection of the Fmoc group releases the aminogroup at the 2′-position to give Compound 45. Compound 45 is treatedwith TBTU(2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate)and triethylamine in DMF to yield Compound 46. Compound 46 isdesilylated with triethylamine trihydrofluoride in triethylamine in THFfollowed by tritylation at 3 position to give the 3-trityloxymethylderivative followed by phosphytilation at 8-position to give Compound47. The DMT phosphoramidite bicyclic nucleoside, Compound 47 is purifiedby silica gel flash column chromatography.

Example 29 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-β-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-β-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl)nucleoside amidites and2′-O-(dimethylaminooxyethyl)nucleoside amidites,2′-(Dimethylaminooxyethoxy)nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([[2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy)nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 30 Oligonucleotide and Oligonucleoside Synthesis

The chimeric oligomeric compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages are prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289,all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 31 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5″-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5″-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3″-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5″-end of the first nucleoside. Thesupport is washed and any unreacted 5″-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5″-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand,. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

RNA oligomeric compounds (RNA oligonucleotides) for use in the presentinvention can be synthesized by the methods herein or purchased fromDharmacon Research, Inc (Lafayette, Colo.). Once synthesized,complementary RNA oligomeric compounds can then be annealed by methodsknown in the art to form double stranded (duplexed) oligomericcompounds. For example, duplexes can be formed by combining 30 μl ofeach of the complementary strands of RNA oligonucleotides (50 uM RNAoligonucleotide solution) and 15 μl of 5× annealing buffer (100 mMpotassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate)followed by heating for 1 minute at 90° C., then 1 hour at 37° C. Theresulting duplexed oligomeric compounds can be used in kits, assays,screens, or other methods to investigate the role of a target nucleicacid.

Example 32 Synthesis of Chimeric Oligomeric Compounds

Chimeric oligomeric compounds, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxyl]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligomeric compounds having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligonucleotide is then recovered by an appropriate method(precipitation, column chromatography, volume reduced in vacuo andanalyzed spetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligomeric compound, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester] Chimericoligomeric compounds

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester] chimericoligomeric compounds are prepared as per the above procedure for the2′-O-methyl chimeric oligomeric compound with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

The above methods are also applicable to the synthesis of chimericoligomeric compounds having multiple alternating regions such asolignucleotides having the formula: T₁-(3′-endo region)-[(2′-deoxyregion)-(3′-endo region)]_(n)-T₂. The use of 2′-MOE or other nucleosideamidites will enable the preparation of a myriad of differentoligonucleotides.

Other chimeric oligomeric compounds, chimeric oligonucleosides and mixedchimeric oligomeric compounds/oligonucleosides are synthesized accordingto U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 33 Screening of Duplexed Oligomeric Compounds of the Invention

In accordance with the present invention, nucleic acid duplexescomprising the oligonucleotides of the invention and their complementsare tested for their ability to modulate the expression of the nucleicacid molecule to which they are targeted. The desired RNA strand(s) ofthe duplex can be synthesized by methods disclosed herein or purchasedfrom various RNA synthesis companies such as for example DharmaconResearch Inc., (Lafayette, Colo.). Once synthesized, the complementarystrands are annealed. The single strands are aliquoted and diluted to aconcentration of 50 uM. Once diluted, 30 uL of each strand is combinedwith 15 uL of a 5× solution of annealing buffer. The final concentrationof the buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2mM magnesium acetate. The final volume is 75 uL. This solution isincubated for 1 minute at 90° C. and then centrifuged for 15 seconds.The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNAduplexes are used in experimentation. The final concentration of thedsRNA compound is 20 uM. This solution can be stored frozen (−20° C.)and freeze-thawed up to 5 times.

Once prepared, the desired synthetic duplexes are evaluated for theirability to modulate target expression. When cells reach approximately60-80% confluency, they are treated with synthetic duplexes comprisingat least one oligomeric compound of the invention. The duplexes aremixed with LIPOFECTIN™ (Invitrogen Life Technologies, Carlsbad, Calif.)in 1 mL of Opti-MEM™-1 reduced serum medium (Invitrogen LifeTechnologies, Carlsbad, Calif.) to achieve the desired finalconcentration of duplex. This transfection mixture was incubated at roomtemperature for approximately 0.5 hours. The final concentration ofduplex ranges from 10 to 200 nM. LIPOFECTIN™ is used at a concentrationof 5 or 6 μg/mL LIPOFECTIN™ per 200 nM of duplex. For cells grown in96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 and thentreated with 130 μL of the transfection mixture. Cells grown in 24-wellplates or other standard tissue culture plates are treated similarly,using appropriate volumes of medium and oligonucleotide. Cells aretreated and data are obtained in duplicate or triplicate. Afterapproximately 4-7 hours of treatment at 37° C., the medium containingthe transfection mixture was replaced with fresh medium. Cells wereharvested 16-24 hours after oligonucleotide treatment, at which time RNAis isolated and target reduction is measured by real-time PCR orNorthern blot.

Example 34 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 35 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 36 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and

UV absorption spectroscopy. The full-length integrity of the individualproducts was evaluated by capillary electrophoresis (CE) in either the96-well format (Beckman P/ACE™ MDQ) or, for individually preparedsamples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI270). Base and backbone composition was confirmed by mass analysis ofthe oligomeric compounds utilizing electrospray-mass spectroscopy. Allassay test plates were diluted from the master plate using single andmulti-channel robotic pipettors. Plates were judged to be acceptable ifat least 85% of the oligomeric compounds on the plate were at least 85%full length.

Example 37 Cell Culture and Oligonucleotide Treatment

The effect of chimeric oligomeric compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or real-time PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal bovine serum (Invitrogen Corporation, Carlsbad, Calif.), 100 unitsper mL penicillin and 100 micrograms per mL streptomycin (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately4000-6000 cells/well for use in oligomeric compound transfectionexperiments.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (Manassas, Va.). A549 cells were routinelycultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum, 100 units per mlpenicillin, and 100 micrograms per ml streptomycin (Invitrogen LifeTechnologies, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of approximately5000 cells/well for use in oligomeric compound transfection experiments.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

HeLa Cells:

The human epitheloid carcinoma cell line HeLa was obtained from theAmerican Tissue Type Culture Collection (Manassas, Va.). HeLa cells wereroutinely cultured in DMEM, high glucose (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal bovine serum (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 90%confluence. Cells were seeded into 24-well plates (Falcon-Primaria#353846, BD Biosciences, Bedford, Mass.) at a density of 50,000cells/well or in 96-well plates (Falcon-Primaria #353872, BDBiosciences, Bedford, Mass.) at a density of 5,000 cells/well for use inoligomeric compound transfection experiments. For Northern blotting orother analyses, cells were harvested when they reached approximately 90%confluence.

NIH3T3 cells:

The mouse embryo-derived NIH3T3 cell line was obtained from AmericanType Culture Collection (Manassas, Va.). NIH3T3 cells were routinelycultured in DMEM, high glucose (Invitrogen Life Technologies, Carlsbad,Calif.) supplemented with 10% fetal bovine serum, (Invitrogen LifeTechnologies, Carlsbad, Calif.), 100 μg/ml penicillin and 100 μg/mlstreptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cellswere routinely passaged by trypsinization and dilution when they reached80% confluencey. Cells were seeded into 96-well plates (Falcon-Primaria#353872, BD Biosciences, Bedford, Mass.) at a density of 3000 cells/wellfor use in oligomeric compound transfection experiments.

b.END Cells:

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Institute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Invitrogen LifeTechnologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum(Invitrogen Life Technologies, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached approximately90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of approximately 3000 cells/well for use inoligomeric compound transfection experiments.

Primary Mouse Hepatocytes:

Primary mouse hepatocytes were prepared from CD-1 mice purchased fromCharles River Labs. Primary mouse hepatocytes were routinely cultured inHepatocyte Attachment Media supplemented with 10% fetal bovine serum, 1%penicillin/streptomycin, 1% antibiotic-antimycotic (Invitrogen LifeTechnologies, Carlsbad, Calif.) and 10 nM bovine insulin (Sigma-Aldrich,St. Louis, Mo.). Cells were seeded into 96-well plates (Falcon-Primaria#3872) coated with 0.1 mg/ml collagen at a density of approximately10,000 cells/well for use in oligomeric compound transfectionexperiments.

Primary Rat Hepatocytes:

Primary rat hepatocytes are prepared from Sprague-Dawley rats purchasedfrom Charles River Labs (Wilmington, Mass.) and are routinely culturedin DMEM, high glucose (Invitrogen Life Technologies, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Life Technologies,Carlsbad, Calif.), 100 units per mL penicillin, and 100 μg/mLstreptomycin (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areseeded into 96-well plates (Falcon-Primaria #353872, BD Biosciences,Bedford, Mass.) at a density of 4000-6000 cells/well for use inoligomeric compound transfection experiments.

MH-S Cells:

The mouse alveolar macrophage cell line was obtained from American TypeCulture Collection (Manassas, Va.). MH-S cells were cultured in RPMIMedium 1640 with L-glutamine (Invitrogen Life Technologies, Carlsbad,Calif.), supplemented with 10% fetal bovine serum, 1 mM sodium pyruvateand 10 mM HEPES (all supplements from Invitrogen Life Technologies,Carlsbad, Calif.). Cells were routinely passaged by trypsinization anddilution when they reached 70-80% confluence. Cells were seeded into96-well plates (Falcon-Primaria #353047, BD Biosciences, Bedford, Mass.)at a density of 6500 cells/well for use in oligomeric compoundtransfection experiments.

Treatment with Oligomeric Compounds:

When cells reached approximately 65-90% confluency, they were treatedwith oligomeric compound. Oligomeric compounds were mixed withLIPOFECTIN™ (Invitrogen Life Technologies, Carlsbad, Calif.) in 1 mL ofOpti-MEM™-1 reduced serum medium (Invitrogen Life Technologies,Carlsbad, Calif.) to achieve the desired concentration of oligomericcompound. The concentration of oligomeric compound used herein rangesfrom 5 to 300 nM. This transfection mixture was incubated at roomtemperature for approximately 0.5 hours. LIPOFECTIN™ is used at aconcentration of 2.5 or 3 μg/mL LIPOFECTIN™ per 100 nM oligomericcompound. For cells grown in 96-well plates, wells were washed once with100 μL OPTI-MEM™-1 and then treated with 130 μL of the transfectionmixture. Cells grown in 24-well plates or other standard tissue cultureplates are treated similarly, using appropriate volumes of medium andoligonucleotide. Cells are treated and data are obtained in duplicate ortriplicate. After approximately 4-7 hours of treatment at 37° C., themedium containing the transfection mixture was replaced with freshmedium. Cells were harvested 16-24 hours after oligonucleotidetreatment, at which time RNA was isolated and target expression wasmeasured by real-time PCR.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 7) which is targeted to human H-ras,or ISIS 18078 (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 8) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are chimericoligomeric compounds composed of a central “gap” segment comprising2′-deoxynucleotides, which is flanked on both sides (5′ and 3′) by“wing” segments comprising 2′-O-methoxyethyl nucleotides(2′-O-methoxyethyls shown in emboldened, underlined type).Internucleoside linkages are phosphorothioate throughout both compounds.All cytosine residues in the wing segments are 5-methylcytosines. Formouse or rat cells the positive control oligonucleotide is ISIS 15770(ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 9) is targeted to both mouse and ratC-raf. ISIS 15770 is a chimeric oligomeric compound composed of acentral “gap” segment comprising 2′-deoxynucleotides, which is flankedon both sides (5′ and 3′) by “wing” segments comprising2′-β-methoxyethyl nucleotides (2′-O-methoxyethyls shown in emboldened,underlined type). Internucleoside linkages are phosphorothioatethroughout the compound. The cytosine residue in the 5′ wing segment isa 5-methylcytosine. The concentration of positive controloligonucleotide that results in 80% inhibition of c-H-ras (for ISIS13920), JNK2 (for ISIS 18078) or C-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of c-H-ras, JNK2 or C-raf mRNA is thenutilized as the oligonucleotide screening concentration in subsequentexperiments for that cell line. If 60% inhibition is not achieved, thatparticular cell line is deemed as unsuitable for oligonucleotidetransfection experiments. The concentrations of antisenseoligonucleotides used herein are from 50 nM to 300 nM.

Example 38 Analysis of Oligonucleotide Inhibition of a Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. One method of RNA analysis of the present invention isthe use of total cellular RNA as described in other examples herein.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Quantitative real-time PCR can beconveniently accomplished using the commercially available ABI PRISM™7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 39 Design of Phenotypic Assays for the Use of Target Inhibitors

Once a target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the target inhibitors. Hallmark genes, or those genes suspected to beassociated with a specific disease state, condition, or phenotype, aremeasured in both treated and untreated cells.

Example 40 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem.,1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation areroutine in the art. Briefly, for cells grown on 96-well plates, growthmedium was removed from the cells and each well was washed with 200 μLcold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 MNaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added toeach well, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 41 Real-Time Quantitative PCR Analysis of a Target mRNA Levels

Quantitation of a target mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time PCR are quantitated as theyaccumulate. This is accomplished by including in the PCR reaction anoligonucleotide probe that anneals specifically between the forward andreverse PCR primers, and contains two fluorescent dyes. A reporter dye(e.g., FAM or JOE, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end ofthe probe and a quencher dye (e.g., TAMRA, obtained from eitherPE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc.,Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa)is attached to the 3′ end of the probe. When the probe and dyes areintact, reporter dye emission is quenched by the proximity of the 3′quencher dye. During amplification, annealing of the probe to the targetsequence creates a substrate that can be cleaved by the 5′-exonucleaseactivity of Taq polymerase. During the extension phase of the PCRamplification cycle, cleavage of the probe by Taq polymerase releasesthe reporter dye from the remainder of the probe (and hence from thequencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

Gene target quantities are obtained by real-time PCR. Prior to thereal-time PCR, isolated RNA is subjected to a reverse transcriptase (RT)reaction, for the purpose of generating complementary DNA (cDNA).Reverse transcriptase and PCR reagents were obtained from InvitrogenCorporation (Carlsbad, Calif.). RT, real-time PCR reactions were carriedout by adding 20 μL PCR cocktail (2.5× PCR buffer minus MgCl₂, 6.6 mMMgCl₂, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forwardprimer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor,1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200ng). The RT reaction was carried out by incubation for 30 minutes at 48°C. Following a 10 minute incubation at 95° C. to activate the PLATINUM®Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for15 seconds (denaturation) followed by 60° C. for 1.5 minutes(annealing/extension).

Gene target quantities obtained by real-time PCR are normalized usingeither the expression level of GAPDH or cyclophilin A, genes whoseexpression levels are constant, or by quantifying total RNA. GAPDHexpression is quantified by real-time PCR, by being run simultaneouslywith the target, multiplexing, or separately. Total RNA is quantifiedusing RiboGreen™ RNA quantification reagent (Molecular Probes, Inc.Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taughtin Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Primers and probes used in real-time PCR are designed with the aid ofcomputer software, for example, Primer Express® Software (PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif.), using publicly available sequence information. It is understoodthat one of skill in the art will readily be able to design such primersand probes.

Example 42 Northern Blot Analysis of a Target mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBONDT™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKERT™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human a target, a human target-specific probe is prepared byPCR. To normalize for variations in loading and transfer efficiencymembranes are stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPhosphorImager™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 43 Western Blot Analysis of a Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPhosphorImager™ (Molecular Dynamics, Sunnyvale Calif.).

Example 44 Gene Target Sequences

In accordance with the present invention, a series of oligomericcompounds was designed to hybridize to different regions of target genesor targets. Presented in Table 12 are the target genes, as well as thecorresponding sequences, identified by GenBank® accession number, usedto design the oligomeric compounds of the invention and other compoundsdescribed herein. “Gene symbol” indicates the name used to herein todescribe the target nucleic acid molecule, and “Gene Name” indicates anadditional name by which the gene target is known.

TABLE 12 Gene target sequences SEQ Gene ID Symbol Gene Name GenBank ®Accession # NO CD86 CD86 S70108.1 10 DGAT2 DiacylglycerolAcyltransferase 2 AK002443.1 11 FAS Fatty Acid Synthase AF127033.1 12FAS Fatty Acid Synthase X62889.1 13 FACL2 Fatty-Acid-Coenzyme A Ligase,Long-Chain 2 NM_007981.1 14 GCGR Glucagon Receptor NM_000160.1 15 GCGRGlucagon Receptor NM_008101.1 16 HSL Hormone-Sensitive Lipase U08188.117 HSD11 Hydroxysteroid 11-Beta Dehydrogenase 1 X83202.1 18 JNK1 JunN-Terminal Kinase-1 L26318.1 19 PP2A- Protein Phosphatase 2 CatalyticSubunit Alpha NM_002715.1 20 alpha PTEN Phosphatase And Tensin HomologueU92436.1 21 PTP1B Protein Tyrosine Phosphatase 1b M33962.1 22 NaDC1Solute Carrier Family 13 (Sodium-Dependent AF201903.1 23 DicarboxylateTransporter), Member 2 SCD1 Stearoyl-Coenzyme A Desaturase 1 1850_038A24 Survivin Survivin U75285.1 25 Survivin Survivin AA717921.1 26Survivin Survivin AB013819.1 27 TRADD Tumor Necrosis Factor ReceptorAssociated L41690.1 28 Death Domain C-raf Raf kinase C X03484.1 29 C-rafRaf kinase C assembled from 30 AC026153.10 and AC018500.2 SRC-2 steroidreceptor coactivator 2 U39060.1 31 SRC-2 steroid receptor coactivator 2complement of nucleotides 32 10220000 to 10460000 of NW_000149.1 SRC-2steroid receptor coactivator 2 AK028964.1 33

Example 45 Chimeric Oligomeric Compounds Having Alternating 3′-Endo and2′-Endo Regions

In one embodiment of the invention, the target sequences presented inTable 12 were used as targets to which oligomeric compounds weredesigned. These compounds have regions of nucleosides that are“RNA-like”, having northern or 3′-endo conformational geometry (3′-endoregions), and regions of nucleosides that are “DNA-like”, havingsouthern or C2′-endo/04′-endo conformational geometry. Each of theregions ranges from 1 to 8 nucleosides in length. The motif of eacholigomeric compound is illustrated in Table 13, where 3′-endo regionsare indicated by bold, underlined type, or by italicized, underlinedtype in the case of ISIS 199043, and 2′-endo regions are indicated byplain type. The number corresponding to each region represents thenumber of base pairs for that particular region. The motif furtherindicates the total number of regions in the compound, for example, acompound having the motif “3-3-1-2-1-2-1-3-4” has a total of 9 regions,with each region ranging from 1 to 4 nucleotides. In the compounds shownin Table 13, the 3′-endo regions shown in bold, underlined type comprise2′-O-methoxyethyl (2′-MOE) nucleotides; the 3′-endo regions initalicized, underlined type comprise 2′-O-methyl nucleotides; and the2′-endo regions comprise 2′-deoxynucleotides. Internucleoside linkagesare phosphorothioate throughout all compound in Table 13, except wherean asterisk “*” is present to indicate a phosphodiester internucleosidelinkage. All cytosines are 5-methylcytosines, unless otherwise indicatedby a superscript “U” preceding the nucleobase, for example, ^(u)C, whichindicates a natural or unmodified cytosine.

The nucleic acid molecule to which each compound is targeted isindicated by SEQ ID NO. “Target site” indicates the first (5′-most)nucleotide number on the particular target nucleic acid to which thecompound binds. Where present, “NA” indicates that “Target SEQ ID NO:”and “Target site” do not apply to a particular oligomeric compound dueto its lack of perfect complementarity to any known gene (i.e., it is amismatched oligomeric compound).

The chimeric oligomeric compounds of the invention, comprising at least5 regions that alternate between 3′-endo regions and 2′-endo regions,are herein referred to as “gap-disabled” oligomeric compounds. Alsodescribed herein are “gapmers”, chimeric oligomeric compounds having 3regions, where one 2′-endo region comprised of 2′-deoxynucleotides isflanked on both sides (5′ and 3′ directions) by a 3′-endo region.

TABLE 13 Oligomeric compounds TARGET SEQ SEQ ID TARGET ID ISIS # NO SITESEQUENCE MOTIF NO 113715  22 980 GCTCC TTCCACTGAT CCTGC 5 -10- 5 45114905  26 296 GTTGG TCTCCTTTGC CTGGA 5 -10- 5 49 116847  21 2097 CTGCTAGCCTCTGGA TTTGA 5 -10- 5 42 118929  20 1492 TCTAC AGTCATGCTG AGTAA 5-10- 5 53 121874  10 289 TCAAG TTTCTCTGTG CCCAA 5 -10- 5 51 121875  10335 GTTCC TGTCAAAGCT CGTGC 5 -10- 5 48 126965  17 2263 CCAGG GCTGCCTCAGACACA 5 -10- 5 39 129605  NA NA CCTGC TCCCTCTAAT GCTGC 5 -10- 5 63129686  NA NA CGTTA TTAACCTCCG TTGAA 5 -10- 5 65 131906  NA NA TCAAGTCCTTCCACA CCCAA 5 -10- 5 70 141923  NA NA CCTTC CCTGAAGGTT CCTCC 5 -10-5 64 146038  18 1107 TTCTC ATGATGAGGT GTACC 5 -10- 5 58 146039  18 1119TGTTG CAAGAATTTC TCATG 5 -10- 5 56 148529  12 630 TTCAT GAACTGCACA GAGGT5 -10- 5 57 148548  12 2238 TTGTT GACATTGTAC TCGGC 5 -10- 5 59 166659 22 980 GCT CCT T CC A CT G ATC CTGC 3 -3- 1 -2- 1 -2- 1 -3- 4 45 180475 16 1348 GAGCT TTGCCTTCTT GCCAT 5 -10- 5 43 189525  NA NA ^(u) C ^(u) CTG^(u) C T^(u)C^(u)C^(u)CT^(u)CTAAT G 5 -10- 5 63 ^(u) CTG ^(u) C 194563 NA NA CC T G CT C C C T C T A A T GCT GC 2 -1- 1 -2- 1 -1- 1 -1- 1 -1-1 - 63 1- 1 -3- 2 199041  NA NA CCTGCTCCCTCTAATGCTGC Uniform 2′-MOE 63199042  NA NA CCTGC TC C CT C TA A T GCTGC 5 -2- 1 -2- 1 -2- 1 -1- 5 63199043  NA NA CCTGCTCCCTCTAATGCTGC Uniform 2′-deoxy 63 199044  NA NACCTGC TCCCTCTAAT GCTGC 5 -10- 5 63 199046  NA NA C*C*T*G*C TCCCTCTAAT G*  5 -10- 5 63 C*T*G*C 199047  NA NA CCTGA TCCCTCTAAT GATGC 5 -10- 5 61199048  NA NA CCTGC TCACTCTAAT GCTGC 5 -10- 5 62 217352  11 1424 ATGCACTCAAGAACT CGGTA 5 -10- 5 35 217376  11 2230 TCCAT TTATTAGTCT AGGAA 5-10- 5 52 244504  24 1329 GTGTT TCTGAGAACT TGTGG 5 -10- 5 47 244541  241435 ATGTC CAGTTTTCCG CCCTT 5 -10- 5 36 249375  23 846 GGACC TGTAGCCATAGCCAA 5 -10- 5 46 249386  23 1021 CTCGT GAACCAGAGC ACCAC 5 -10- 5 41256899  13 12343 TTGTT GACGTTGTAC TCAGC 5 -10- 5 60 283586  22 980GCTCCTTCCACTGATCCTGC Uniform 2′-MOE 45 284346  NA NA CTTCT AGCCTCTGGATTGGA 5 -10- 5 66 291452  14 214 TCAAG GACTGCTGAT CTTCG 5 -10- 5 50298682  NA NA GCGAT TTCCCGTTTT CACCT 5 -10- 5 67 298683  16 1348GAGCTTTGCCTTCTTGCCAT Uniform 2′-MOE 43 298683 16 1348GAGCTTTGCCTTCTTGCCAT Uniform 2′-MOE 43 299228 25 12665 TG TG C TAT T CTG TGA ATT 2 -2- 1 -3- 1 -2- 1 -3- 3 55 299229 25 12665 TGT GCT A TT CTGT G AA TT 3 -3- 1 -2- 1 -3- 1 -2- 2 55 299230 27 856 AAC CA C ACT T ACC CAT GGGC 3 -2- 1 -3- 1 -2- 1 -3- 4 34 299231 26 296 GTT GG T CTC C TTT GCC TGGA 3 -2- 1 -3- 1 -2- 1 -3- 4 49 299232 27 303 TGT CA T CGG G TTC CCA GCCT 3 -2- 1 -3- 1 -2- 1 -3- 4 54 300861 16 1348 GAG CT T TGC CTTC T TGC CAT 3 -2- 1 -3- 1 -3- 1 -3- 3 43 303767 NA NA GTTCG TGTTCTCTGGCTCGA 5 -10- 5 68 304170 13 12343 TTG TT G AC G TT G TA C TC AGC 3 -2- 1-2- 1 -2- 1 -2- 1 -2- 3   60 304171 12 2238 TTG TT G AC A TT G TA C TCGGC 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   59 306058 10 289 TCA AG T TT C TCTG TG C C CAA 3 -2- 1 -2- 1 -3- 1 -2- 1 -1- 3   51 307754 19 341 ATTTGCATCCATGAG CTCCA 5 -10- 5 37 310456 15 500 CAGGA GATGTTGGCC GTGGT 5 -10-5 38 310457 15 532 GCACT TTGTGGTGCC AAGGC 5 -10- 5 44 310514 11 1424 ATGCA C TC A AG A AC T CG GTA 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   35 31051511 2230 TCC AT T TA T TA G TC T AG GAA 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3  52 310516 18 1107 TTC TC A TG A TG A GG T GT ACC 3 -2- 1 -2- 1 -2- 1 -2-1 -2- 3   58 310517 18 1119 TGT TG C AA G AA T TT C TC ATG 3 -2- 1 -2- 1-2- 1 -2- 1 -2- 3   56 312837 23 846 GGA CC T GT A GC C AT A GC CAA 3-2- 1 -2- 1 -2- 1 -2- 1 -2- 3   46 312844 24 1329 GTG TT T CT G AG A ACT TG TGG 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   47 319162 14 214 TCA AG G ACT GC T GA T CT TCG 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   50 319237 NA NATTGTT AACGGTGTTC TCAGC 5 -10- 5 71 319238 NA NA TTTGT AACGGTGTTC ACTGA 5-10- 5 72 319239 12 630 TTC AT G AA C TG C AC A GA GGT 3 -2- 1 -2- 1 -2-1 -2- 1 -2- 3   57 319240 NA NA TACTT GACCTACAGA GTGGA 5 -10- 5 69330693 17 2263 CC A G GG C T G C C T C A G ACA CA 2 -1- 1 -2- 1 -1- 1-1- 1 -1- 1 - 39 1- 1 -3- 2 332520 15 532 GCACTTTGTGGTGCCAAGGCUniform 2′-MOE 44 332521 15 532 GCACTTTGTGGTGCCAAGGC Uniform 2′-deoxy 44332522 15 532 GCA CT T TG T GG T GC C AA GGC 3 -2- 1 -2- 1 -2- 1 -2- 1-2- 3   44 332864 16 1348 GAGC TTT G CCTT C TTG CCAT 4 -3- 1 -4- 1 -3- 443 332865 16 1348 GAG CT T TG C CT T CT T GC CAT 3 -2- 1 -2- 1 -2- 1 -2-1 -2- 3   43 332866 16 1348 GAG CTTTG CCTT CTTGC CAT 3 -5- 4 -5- 3 43332867 16 1348 GAG CTTTGCCTTCTTGC CAT 3 -14- 3 43 332868 16 1348 GAG CTTTG CCTT CT TGC CAT 3 -3- 2 -4- 2 -3- 3 43 332869 16 1348 GAG C T T T G CC T T C T T G CCAT 3 -1- 1 -1- 1 -1- 1 -1- 1 -1- 1 - 43 1- 1 -1- 4333022 15 500 CAGGAGATGTTGGCCGTGGT Uniform 2′-MOE 38 333023 15 500CAGGAGATGTTGGCCGTGGT Uniform 2′-deoxy 38 333024 15 500 CAG GA G AT G TTG GC C GT GGT 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   38 334269 21 2097 CTGCTAGCCTCTGGATT TGA 3 -14- 3 42 334270 21 2097 CTG CTAGCC T CTGGATT TGA 3-6- 1 -7- 3 42 334271 21 2097 CTG CTAGCCT C TGGATT TGA 3 -7- 1 -6- 3 42334272 21 2097 CTG CTAGCCTCT G GATT TGA 3 -4- 1 -4- 1 -4- 3 42 334273 212097 CTG CTA G CC T CTG G ATTTGA 3 -3- 1 -2- 1 -3- 1 -3- 3 42 334274 212097 CTG CTA G CCT C TG G ATT TGA 3 -3- 1 -3- 1 -2- 1 -3- 3 42 334275 212097 CTG CT A GC C TC T GG A TT TGA 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   42334276 21 2097 C*T*G*C*T*A*G*C*C*T* Uniform 2′-deoxy 42C*T*G*G*A*T*T*T*G*A 335032 16 1348 GAGCTTTGCCTTCTTGCCAT Uniform 2′-deoxy43 335033 16 1348 G*A*G*C*T*T*T*G*C*C* Uniform 2′-deoxy 43T*T*C*T*T*G*C*C*A*T 335112 16 1348 G * A * G * C * T *T*T*G*C*C* 5 -10-5 43 T*T*C*T*T* G * C * C * A * T 335114 16 1348 G * A * G *C*T* T*T*G*C* C * 3 -2- 1 -3- 1 -3- 1 -3- 3 43 T*T*C* T *T*G*C* C * A * T337205 11 1424 ATG CACTCAAGAACTCG GTA 3 -14- 3 35 337206 11 1424 ATGCACTCA A GAACTCG GTA 3 -6- 1 -7- 3 35 337207 11 1424 ATG CACTCAA GAACTCG GTA 3 -7- 1 -6- 3 35 337208 11 1424 ATG CACT C AAGA A CTCG GTA 3-4- 1 -4- 1 -4- 3 35 337209 11 1424 ATG CAC T CA A GAA C TCG GTA 3 -3- 1-2- 1 -3- 1 -3- 3 35 337210 11 1424 ATG CAC T CAA G AA C TCG GTA 3 -3- 1-3- 1 -2- 1 -3- 3 35 337211 11 1424 A*T*G*C*A*C*T*C*A*A*Uniform 2′-deoxy 35 G*A*A*C*T*C**G*G*T*A 337212 11 1424 ATG CA CT C AA GAA C T CG GTA 3 -2- 2 -1- 2 -1- 2 -1- 1 -2- 3   35 337213 11 1424 ATG CACT C AA G AA C TC G GTA 3 -1- 3 -1- 2 -1- 2 -1- 2 -1- 3   35 337214 111424 ATG C AC T CA A GA A CT C GGTA 3 -1- 2 -1- 2 -1- 2 -1- 2 -1- 4   35337215 11 1424 ATG C A C T C A A G A A C T C GGTA 3 -1- 1 -1- 1 -1- 1-1- 1 -1- 1 - 35 1- 1 -1- 4 337216 11 1424 ATG C A C T C A A G A A C T CG G TA 1 -1- 1 -1- 1 -1- 1 -1- 1 -1- 1 - 35 1- 1 -1- 1 -1- 1 -1- 2337217 21 2097 CTG CT AG C CT C TG G A TT TGA 3 -2- 2 -1- 2 -1- 2 -1- 1-2- 3   42 337218 21 2097 CTG C TAG C CT C TG G AT T TGA 3 -1- 3 -1- 2-1- 2 -1- 2 -1- 3   42 337219 21 2097 CTG C TA G CC T CT G GA T TTGA 3-1- 2 -1- 2 -1- 2 -1- 2 -1- 4   42 337220 21 2097 CTG C T A G C C T C TG G A T TTGA 3 -1- 1 -1- 1 -1- 1 -1- 1 -1- 1 - 42 1- 1 -1- 4 337221 212097 C T G C T A G C C T C T G G A T T T GA 1 -1- 1 -1- 1 -1- 1 -1- 1-1- 1 - 42 1- 1 -1- 1 -1- 1 -1- 2 337222 11 1424 ATGCACTCAAGAACTCGGTAUniform 2′-MOE 35 338173 28 802 CGCTC GTACTCGTAG GCCAG 5 -10- 5 40338174 28 802 CGCTCGTACTCGTAGGCCAG Uniform 2′-MOE 40 338175 28 802 CGCTCGT A CTCG T AGG CCAG 4 -3- 1 -4- 1 -3- 4 40 338176 28 802 CGC TC G TA CTC G TA G GC CAG 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   40 338177 28 802 CGCTCGTA CTCG TAGGC CAG 3 -5- 4 -5- 3 40 338178 28 802 CGC TCGTACTCGTAGGCCAG 3 -14- 3 40 338179 28 802 CGC TCG TA CTCG TA GGC CAG 3 -3- 2 -4- 2-3- 3 40 338180 28 802 CGC T C G T A C T C G T A G G CCAG 3 -1- 1 -1- 1-1- 1 -1- 1 -1- 1 - 40 1- 1 -1- 4 345888 19 341 ATT TG C AT C CA T GA GCT CCA 3 -2- 1 -2- 1 -2- 1 -2- 1 -2- 3   37 352426 16 1348 GA GCTTTGCCTT CTTGCC AT 2 -6- 4 -6- 2 43 352427 16 1348 GA GCTTTGC CT TCTTGCC AT2 -7- 2 -7- 2 43 352428 16 1348 G AGCTTTGC CT TCTTGCCA T 1 -8- 2 -8- 143

The target regions to which these sequences are complementary are hereinreferred to as “target segments” and are therefore suitable fortargeting by oligomeric compounds of the present invention. The targetsegment sequences represent the reverse complement of the chimericoligomeric compounds.

As these “target segments” have been found by experimentation to be opento, and accessible for, hybridization with the chimeric oligomericcompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother oligomeric compounds that specifically hybridize to these targetsegments and consequently inhibit the expression of a target.

According to the present invention, chimeric oligomeric compoundsinclude antisense oligomeric compounds, antisense oligonucleotides,siRNAs, alternate splicers and other short oligomeric compounds whichhybridize to at least a portion of the target nucleic acid.

Example 46 In Vitro Analysis of Chimeric Oligomeric Compounds HavingAlternating 3′-Endo and 2′-Endo Regions

In one embodiment, gap-disabled oligomeric compounds were selected fromTable 13 and tested for their effects on target expression in culturedcells. Gapmer compounds were also tested in each in vitro assay andserved as the positive control for target reduction.

To test the effects of gap-disabled compounds of the invention on mousesurvivin expression, NIH 3T3 cells were treated 6.25, 25, 100 and 200 nMof the oligomeric compounds shown in Table 13. ISIS 303767, whichcontains 6 mismatches to mouse survivin, was used as a negative controlin this assay. Cells were transfected using LIPOFECTINT™ and mRNA levelswere measured using real-time PCR as described in other examples herein.Results of these studies are shown in Table 14. Data are averages fromtwo or more experiments and are expressed as percent inhibition relativeto untreated control. As demonstrated in Table 14, the gap-disabledcompounds ISIS 299230 and ISIS 229231 and the gapmer ISIS 114905inhibited mouse survivin expression in a dose-dependent manner. Thegap-disabled compounds ISIS 299229 and ISIS 299232 inhibited mousesurvivin expression at the 100 and 200 nM doses.

TABLE 14 Inhibition of mouse survivin expression in NIH 3T3 cells: doseresponse % Inhibition SEQ ID Dose of oligonucleotide (nM) ISIS # NO 6.2525 100 200 299228 55 0 0 0 9 299229 55 0 0 10 17 299230 34 23 28 64 72299231 49 22 44 78 83 299232 54 0 0 38 59 114905 49 0 51 82 91 303767 680 0 10 60

Oligomeric compounds targeting mouse SCD1 were also tested. Primarymouse hepatocytes were treated with 15, 44, 133 and 400 nM of theoligomeric compounds shown in Table 15, or the control oligomericcompound ISIS 141923, which does not target mouse SCD1. Cells weretransfected using LIPOFECTINT™ and mRNA levels were measured usingreal-time PCR as described in other examples herein. Results of thesestudies are shown in Table 15. Data are averages from three experimentsand are expressed as percent inhibition relative to untreated control.As demonstrated in Table 15, the gap-disabled compound ISIS 312844inhibited SCD1 expression in a dose-dependent manner. The gapmercompounds also inhibited SCD1 expression in a dose-dependent manner.

TABLE 15 Inhibition of mouse SCD1 expression in mouse primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nM) ISIS # NO 15 44 133 400 312844 47 0 15 40 69 244504 47 15 32 65 83244541 36 0 1 46 78 141923 64 0 0 0 0

To evaluate the effects of oligomeric compounds of the invention onmouse PTEN expression, b.END cells were treated with 12.5, 25, 50 or 100nM of the oligomeric compounds shown in Table 16. ISIS 141923, whichdoes not target PTEN, was used as a negative control in this assay.Cells were transfected using LIPOFECTINT™ and mRNA levels were measuredusing real-time PCR as described in other examples herein. Results ofthese studies are shown in Table 16. Data are averages from two or moreexperiments and are expressed as percent inhibition relative tountreated control. As demonstrated in Table 16, the gap-disabledcompounds ISIS 334269, ISIS 334270, ISIS 334271, ISIS 334272, ISIS334273, ISIS 334274 and ISIS 334275 inhibited mouse PTEN mRNA expressionin a dose-dependent manner, as did the gapmer compound ISIS 116847. ISIS334269, a gapmer compound with a gap segment 14 nucleotides in lengthand wing segments 3 nucleotides in length, also inhibited PTENexpression in a dose-dependent manner. The uniform 2′-deoxy compoundISIS 334276 did not exhibit target inhibition greater than 9%.

TABLE 16 Inhibition of mouse PTEN mRNA expression in b.END cells: doseresponse % Inhibition SEQ ID Dose of oligonucleotide (nM) ISIS # NO 12.525 50 100 334269 42 9 29 56 71 334270 42 31 29 63 75 334271 42 18 46 5966 334272 42 0 31 57 64 334273 42 19 31 47 60 334274 42 9 26 47 50334275 42 10 30 43 63 334276 42 3 9 8 0 116847 42 12 45 62 75 141923 640 0 0 0

Additional compounds targeted to mouse PTEN were tested in a similarassay in b.END cells. Cells were transfected using LIPOFECTINT™ and mRNAlevels were measured using real-time PCR as described in other examplesherein. ISIS 337217 inhibited target expression 10% and 15% at doses of25 and 50 nM, respectively. ISIS 331218 inhibited PTEN expression by 17%at a dose of 100 nM. ISIS 337219, ISIS 337220 and ISIS 337221 did notsignificantly inhibit PTEN expression in b.END cells in this assay.

Oligomeric compounds targeted to NaDC1 were also tested in an in vitroassay. Primary mouse hepatocytes were treated with 15, 44, 133 or 400 nMof the oligomeric compounds shown in Table 17. ISIS 141923, which doesnot target mouse NaDC1, was used as a negative control compound in thisassay. Cells were transfected using LIPOFECTINT™ and mRNA levels weremeasured using real-time PCR as described in other examples herein.Results of these studies are shown in Table 17. Data are averages fromthree experiments and are expressed as percent inhibition relative tountreated control. As demonstrated in Table 17 the gap-disabled compoundISIS 312387 inhibited mouse NaDC1 in a dose-dependent manner, as did thegapmer compounds targeted to NaDC1.

TABLE 17 Inhibition of mouse NaDC1 mRNA expression in mouse primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nM) ISIS NO NO 15 44 133 400 312837 46 16 55 61 79 249375 46 29 59 7190 249386 41 0 9 38 76 141923 64 0 0 0 0

Primary mouse hepatocytes were treated for 4 hours with 15, 44, 133, and400 nM of the oligomeric compounds shown in Table 18. ISIS 141923, whichdoes not target mouse HSD11, was used as a negative control in thisassay. Cells were transfected using LIPOFECTINT™ and mRNA levels weremeasured using real-time PCR as described in other examples herein.Results of these studies are shown in Table 18. Data are averages fromthree experiments and are expressed as percent inhibition relative tountreated control. As demonstrated in Table 18, the gap-disabledcompound ISIS 310516 inhibited HSD11 expression in a dose-dependentmanner, as did the gapmer compound.

TABLE 18 Inhibition of mouse HSD11 mRNA expression in mouse primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nM) ISIS NO NO 15 44 133 400 310516 58 0 40 69 95 146038 58 37 70 94 97141923 64 0 0 0 0

Gap-disabled compound targeting the mouse glucagon receptor RNA werealso tested in an in vitro assay. Primary mouse hepatocytes were treatedwith 0.5, 1, 5, 10, 25 or 50 nM of the oligomeric compounds shown inTable 19. ISIS 116847, which does not target the mouse glucagonreceptor, was used as a negative control in this assay. Cells weretransfected using LIPOFECTINT™ and mRNA levels were measured usingreal-time PCR as described in other examples herein.

Results of these studies are shown in Table 19. Data are averages fromthree experiments and are expressed as percent inhibition relative tountreated control. “IC₅₀” indicates the concentration of oligomericcompound required to inhibit glucagon receptor mRNA expression by 50%.Where present, “ND” indicates “not determined.” As demonstrated in Table19, the gap-disabled compounds ISIS 300861, ISIS 332864, ISIS 332865,ISIS 332866, ISIS 332897 and ISIS 332868 inhibited mouse glucagonreceptor expression in a dose-dependent manner, as did the gapmercompound. ISIS 332867, a gap-disabled compound, inhibited mouse glucagonreceptor expression. ISIS 332869, a gapmer compound with a gap segmentof 14 nucleotides in length and wing segments of 3 nucleotides inlength, exhibited dose-dependent inhibition of mouse glucagon receptormRNA at doses of 5, 10 and 25 nM.

TABLE 19 Inhibition of mouse glucagon receptor mRNA expression in mouseprimary hepatocytes: dose response % Inhibition SEQ ID Dose ofoligonucleotide (nM) IC₅₀ ISIS # NO 0.5 1 5 10 25 50 (nM) 300861 43 1 1532 20 51 67 24 332864 43 10 30 52 45 63 70 14 332865 43 6 10 29 36 49 5333 332866 43 27 42 ND 58 70 75 6 332867 43 37 48 66 74 74 77 1 332868 437 34 52 58 68 ND 5 332869 43 15 2 5 12 24 25 >50 180475 43 3 43 58 68 7880 3 116847 42 11 13 0 0 0 0 >50

To evaluate the effects of gap-disabled compounds targeted to mouseDGAT2, primary mouse hepatocytes were treated with 15, 44, 133, and 400nM of the oligomeric compounds shown in Table 20. ISIS 116847, whichdoes not target the mouse glucagon receptor, was used as a negativecontrol in this assay. Cells were transfected using LIPOFECTINT™ andmRNA levels were measured using real-time PCR as described in otherexamples herein. Results of these studies are shown in Table 20. Dataare averages from three experiments and are expressed as percentinhibition relative to untreated control. As demonstrated in Table 2 thegap-disabled compounds ISIS 310514 and ISIS 310515, like the gapmercompounds, inhibited mouse DGAT2 expression in a dose-dependent manner.

TABLE 20 Inhibition of mouse DGAT2 expression in mouse primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nM) ISIS NO NO 15 44 133 400 310514 35 32 64 78 88 310515 52 0 39 45 66217352 35 71 87 94 95 217376 52 65 75 91 98 141923 64 43 44 0 0

An additional assay tested a gap-disabled compound targeted to mouseCD86. MH-S cells were treated with 0.12, 0.37, 1, 1, 3.3, 10 and 30 nMof the oligomeric compounds shown in Table 21. ISIS 131906, whichcontains seven mismatched bases to mouse CD86, served as the negativecontrol compound in this assay. Cells were transfected usingLIPOFECTINT™ and mRNA levels were measured using real-time PCR asdescribed in other examples herein. Data are averages from two or moreexperiments and are expressed as percent inhibition relative tountreated control. Results of these studies are shown in Table 21 anddemonstrate that the gap-disabled compound ISIS 306058, inhibited CD86mRNA expression in a dose-dependent manner at doses of 3.3, 10 and 30nM.

TABLE 21 Inhibition of mouse CD86 mRNA expression in MH-S cells: doseresponse % Inhibition SEQ ID Dose of oligonucleotide (nM) ISIS # NO 0.120.37 1.1 3.3 10 30 306058 51 0 6 0 17 32 45 121874 51 0 27 42 61 74 70121875 48 0 25 43 62 78 81 131906 70 0 1 0 0 0 21

In a further embodiment, ISIS 306058 was tested for its ability tomodulate cell surface expression of CD86 protein. MH-S cells weretreated with 0.1, 0.4, 1.2, 3.7, 11.1, 33.3 and 100 nM of the oligomericcompounds shown in Table 22. ISIS 131906, which contains sevenmismatched bases to mouse CD86, served as the negative control compoundin this assay. Cells were transfected using LIPOFECTINT™ as described inother examples herein. Cell surface expression of CD86 protein wasmeasured by flow cytometry. Cell surface expression of CD80, whichshares sequence identify with CD86 at the nucleic acid level, was alsomeasured. Cells were harvested by brief trypsinization, washed with PBS,then resuspended in 100 μL of staining buffer (PBS, 0.2% BSA) containingboth 10 μL of FITC-conjugated anti-CD86 antibody (FITC-anti-hCD86; FITC:fluorescien isothiocyanate; BD Biosciences, San Jose, Calif.) and 10 ulof PE-conjugated anti-CD80 antibody (PE: phycoerythrin; PE-anti-hCD80,BD Biosciences, San Jose, Calif.). The cells were stained for 30 minutesat 4° C., washed with PBS, resuspended in 300 μL PBS containing 0.5%paraformaldehyde. Measurements of mean fluorescence activity were madeby flow cytometry using the FL-1 and FL-2 channels of a BD BiosciencesFACScan (BD Biosciences, San Jose, Calif.). With this method, both CD86and CD80 protein expression on the surface of the same cell wasmeasured. Data were averaged from two or more experiments and areexpressed as percent inhibition relative to untreated control. As shownin Table 22, the gap-disabled compound ISIS 306058 exhibited inhibitionof CD86 protein expression in a pattern similar to that observed incells treated with the gapmer compounds, with dose-dependent inhibitionlimited to the 5 lower doses. CD80 protein levels were not lowered bythe gap-disabled or gapmer compounds targeted to CD86.

TABLE 22 Inhibition of mouse CD86 protein expression in MH-S cells: doseresponse % Inhibition SEQ ID Dose of oligonucleotide (nM) ISIS # NO 0.10.4 1.2 3.7 11.1 33.3 100 306058 51 4 6 12 19 30 31 31 121874 51 10 2243 56 57 57 57 121875 48 9 21 34 46 57 55 54 131906 70 0 6 9 3 3 15 30

A gap-disabled compound targeted to mouse ACS1 was tested for itseffects on target mRNA expression. Primary mouse hepatocytes weretreated with 15, 44, 133 and 400 nM of the oligomeric compounds shown inTable 23. ISIS 141923, which does not target mouse ACS1, was used as anegative control in this assay. Cells were transfected usingLIPOFECTINT™ and mRNA levels were measured using real-time PCR asdescribed in other examples herein. Data were averaged from threeexperiments and are expressed as percent inhibition relative tountreated control. Results of these studies are shown in Table 23 anddemonstrate that the gap-disabled compound ISIS 319962 inhibited mouseACS1 in a dose-dependent manner, as the gapmer compound.

TABLE 23 Inhibition of mouse ACS1 expression in mouse primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nM) ISIS # NO 15 44 133 400 319162 50 9 12 45 77 291452 50 20 38 63 90141923 64 32 5 17 29

An additional in vitro assay was performed to test a gap-disabledcompound targeted to rat HSD11. Primary rat hepatocytes were treated for4 hours with 15, 44, 133 and 400 nM of the oligomeric compounds shown inTable 24. ISIS 141923, which does not target rat HSD11, was used as anegative control in this assay. Cells were transfected usingLIPOFECTINT™ and mRNA levels were measured using real-time PCR asdescribed in other examples herein. Data were averaged from threeexperiments and are expressed as percent inhibition relative tountreated control. Results of these studies are shown in Table 24 anddemonstrate that the gap-disabled compound ISIS 310517 inhibited targetmRNA expression in a dose-dependent manner at the 3 higher doses ofoligomeric compound.

TABLE 24 Inhibition of rat HSD11 mRNA expression in rat primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nM) ISIS NO NO 15 44 133 400 146039 56 31 53 76 92 310517 56 0 20 54 79141923 64 7 9 0 0

Gap-disabled compounds targeted to rat FAS were tested for their effectson target mRNA expression. Primary rat hepatocytes were treated with 5,10, 25, 50, 100, and 200 nM of the oligomeric compounds shown in Table25. ISIS 319237, ISIS 319238, or ISIS 319240, which contain 3, 8 and 7mismatches to rat FAS, respectively, were used as negative controlcompounds in this assay. Cells were transfected using LIPOFECTINT™ andmRNA levels were measured using real-time PCR as described in otherexamples herein. Results of these studies are shown in Table 25. Dataare averages from three experiments and are expressed as percentinhibition relative to untreated control. “IC₅₀” indicates theconcentration of oligomeric compound required to inhibit FAS mRNAexpression by 50%. Where present, “ND” indicates “not determined.” Thedata illustrate that the gap-disabled compound ISIS 304170 inhibited ratFAS mRNA in a dose-dependent manner. With the exception of the 25 nMdose, the treatments with ISIS 319239 inhibited rat FAS expression in adose-dependent manner. The gapmer compounds also inhibited targetexpression, whereas the mismatched compounds did not.

TABLE 25 Inhibition of rat FAS mRNA expression in rat primaryhepatocytes: dose response % Inhibition SEQ ID Dose of oligonucleotide(nm) IC₅₀ ISIS # NO 5 10 25 50 100 200 (nM) 304170 60 1 12 9 13 42 71104 319239 57 4 14 0 38 62 76 67 148529 57 0 0 8 28 61 70 75 256899 6017 16 3 25 52 73 97 319237 71 0 0 0 0 0 5 N.D. 319238 72 0 0 0 0 0 0N.D. 319240 69 0 0 0 0 0 13 N.D.

From the data from the in vitro assays presented in Tables 14-25, it isevident that gap-disabled compounds effectively inhibited the expressionof the nucleic acid molecules to which they are targeted.

Example 47 Chimeric Oligomeric Gap-Disabled Compounds Having Varying 2′Sugar Modifications

The data described herein demonstrate that gap-disabled oligomericcompounds having 2′-MOE nucleotides in the 3′-endo regions are able toinhibit expression of a target gene. In a further embodiment, a seriesof oligomeric compounds was designed, using various 2′ sugarmodifications in the 3′-endo region. The oligomeric compounds weredesigned using SEQ ID NO: 43, which targets the mouse glucagon receptorRNA. The compounds are shown in Table 26. All compounds in Table 26 arechimeric oligomeric compounds comprising regions that alternate between3′-endo regions and 2′-endo regions. The motif of each oligomericcompound is illustrated in Table 26, where 3′-endo regions are indicatedby bold, underlined type and 2′-endo regions are indicated by plaintype. The number corresponding to each region represents the number ofbase pairs for that particular region. The 3′-endo modification of eacholigomeric compound is also indicated in Table 26. All internucleosidelinkages are phosphorothioate throughout each compound in Table 26.Unmodified cytosines are indicated by a superscript “U” preceding thenucleobase, for example, “^(U)C”; all other cytosines are5-methylcytosines. The 2′-endo regions of ISIS 340662 are comprised of2′-ribonucleotides. The 2′-endo regions of all other compounds in Table26 are comprised of 2′-deoxynucleotides. Where indicated by “U” at the3′-terminal nucleobase position of ISIS 340658, ISIS 340661, ISIS 340663and ISIS 358699, uracil was used in place of thymidine, making thecompounds hybrids of DNA and RNA.

TABLE 26 Gap-disabled oligomeric compounds targeted to mouseglucagon receptor: varying motifs and 3′-endo nucleosides SEQ ISIS ID3′-endo NO NO Sequence (5′ to 3′) Motif modification 180475 43 GAGCTTTGCCTTCTT GCCAT 5 -10- 5 2′-MOE 298683 43 GAGCTTTGCCTTCTTGCCATUniform 2′-MOE 2′-MOE 300861 43 GAG CT T TGC C TTC T TGC CAT 3 -2- 1 -3-1 -3- 1 -3- 3   2′-MOE 340658 43 GAG CT T TGC C TTC T TGC CAU 3 -2- 1-3- 1 -3- 1 -3- 3   2′-O-methyl 340659 43 GAG CT T TGC C TTC T TGC CAT 3-2- 1 -3- 1 -3- 1 -3- 3   2′-fluoro 340660 43 GAG CT T TGC C TTC T TGCCAT 3 -2- 1 -3- 1 -3- 1 -3- 3   LNA 340661 43 GAG CT U TGC^(U) C TTC UTGC^(U) CAU 3 -2- 1 -3- 1 -3- 1 -3- 3   2′-OH 340662 43 GAG ^(U)CU TUG^(U)C C UU^(U)C T UG^(U)C CAT    3 -2- 1 -3- 1 -3- 1 -3- 3 2′-MOE332866 43 GAG CTTTG CCTT CTTGC CAT 3 -5- 4 -5- 3 2′-MOE 340663 43 GAGCTTTG CCTT CTTGC CAU 3 -5- 4 -5- 3 2′-O-methyl 340673 43 GAG CTTTG CCTTCTTGC CAT 3 -5- 4 -5- 3 LNA 358699 43 GAG CTTTG CCTT CTTGC CAU 3 -5- 4-5- 3 2′-fluoro

The compounds were tested for their ability to modulate the expressionof glucagon receptor mRNA in mouse primary hepatocytes. Cells, culturedas described herein, were treated with 0.1, 0.316, 1, 3.16, 10, 31.6 or100 nM of oligomeric compounds. Untreated cells served as a controlgroup to which all other data were normalized. Cells were transfectedand mRNA was measured as described herein. The data, shown in Table 27,are the average of 3 experiments and are presented as percent of controlcell mRNA expression. A number less than or greater than 100% indicatesa decrease or increase in mRNA expression, respectively.

TABLE 27 Oligomeric compounds of varying motifs and 3′-endo regions:effects on mouse glucagon receptor mRNA Dose of oligomeric compound (nM)100 31.6 10 3.16 1 0.316 0.1 3′-endo ISIS # % Control expression Motifmodification 180475 16 58 84 130 140 141 103 5-10-5 2′-MOE 298683 105133 149 167 150 133 144 Uniform 2′-MOE 2′-MOE 300861 58 109 116 145 151162 132 3-2-1-3-1-3-1-3-3 2′-MOE 340658 78 100 131 141 171 160 1193-2-1-3-1-3-1-3-3 2′-O-methyl 340659 62 85 118 131 138 154 1343-2-1-3-1-3-1-3-3 2′-fluoro 340660 38 61 97 121 134 146 1543-2-1-3-1-3-1-3-3 LNA 340661 93 129 129 124 165 146 1163-2-1-3-1-3-1-3-3 2′-OH 340662 99 151 145 149 163 168 1283-2-1-3-1-3-1-3-3 2′-MOE 332866 20 64 83 146 133 128 144 3-5-4-5-32′-MOE 340663 25 76 112 123 137 138 137 3-5-4-5-3 2′-O-methyl 340673 4559 87 112 128 125 99 3-5-4-5-3 LNA 358699 42 75 113 118 158 115 1473-5-4-5-3 2′-fluoro

These data demonstrate that gap-disabled compounds, having a pluralityof motifs and 3′-endo modifications, exhibit target reduction activityin this assay. For example, ISIS 300861 (2′-MOE), ISIS 340658(2′-O-methyl), ISIS 340659 (2′-fluoro), ISIS 340660 (LNA), ISIS 332866(2′-MOE), ISIS 340663 (2′-O-methyl), ISIS 340673 (LNA) and ISIS 359699(2′-fluoro) inhibited target expression at the 100 nM dose.

Example 48 Comparison of Gapmers and Gap-Disabled Oligomeric Compounds:Influence on Apoptosis Induction and Cell Viability

Programmed cell death, or apoptosis, is an important aspect of variousbiological processes, including normal cell turnover, as well as immunesystem and embryonic development. Apoptosis involves the activation ofcaspases, a family of intracellular proteases through which a cascade ofevents leads to the cleavage of a select set of proteins. The caspasefamily can be divided into two groups: the initiator caspases, such ascaspase-8 and -9, and the executioner caspases, such as caspase-3, -6and -7, which are activated by the initiator caspases. The caspasefamily contains at least 14 members, with differing substratepreferences (Thornberry and Lazebnik, Science, 1998, 281, 1312-1316).Measuring caspase-3 activity is one manner in which caspase activity isevaluated. Changes in nucleic acid content also serve as an indicator ofcell viability, as well as cytotoxic events or pathologicalabnormalities that affect cell proliferation.

The ability of gap-disabled and gapmer oligomeric compounds to affectapoptosis and viability in cultured cells was assayed using gap-disabledcompounds and their corresponding gapmer compounds. The nucleic acidmolecules to which these compounds are targeted, as well as the sequenceand motif of each compound, are shown in Table 13. The gap-disabledcompounds were: ISIS 330693, ISIS 194563, ISIS 300861 and ISIS 304170.The gapmer compounds were: ISIS 126965, ISIS 129605, ISIS 180475 andISIS 256899.

These were tested for their effects on caspase-3 activity and cellviability in the human lung carcinoma cell line A549 (American TypeCulture Collection; Manassas, Va.). A549 cells were routinely culturedin DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.)supplemented with 10% fetal bovine serum (Invitrogen Corporation,Carlsbad, Calif.) and lx antibiotic-antimycotic mix (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached approximately 100%confluence. For LIPOFECTINT™-mediated transfection A549 cells wereplated on 96-well microtiter plates (Falcon-Primaria #353872, BDBiosciences, Bedford, Mass.) precoated with rat tail collagen (BDBiosciences, Bedford Mass.) at a density of approximately 2*10⁵ cells/mlin Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovineserum and antibiotic-antimycotic mix. Cells were cultured overnight at37° C. in the presence of 5% CO₂. The following day the media wasaspirated and replaced with prewarmed OPTI-MEM™ (Invitrogen Corporation,Carlsbad, Calif.) containing 300 nM oligonucleotide and 9 μg/mLLIPOFECTINT™ (Invitrogen Corporation, Carlsbad, Calif.). Cells incubatedwith OPTI-MEM™ alone served as untreated control cells. After four hoursthe transfection mix was exchanged for fresh culture medium and cellswere incubated for an additional 44 hours at 37° C. in the presence of5% CO₂.

Caspase-3 activity was evaluated with a fluorometric HTS Caspase-3 assay(Oncogene Research Products, San Diego, Calif.) that detects cleavageafter aspartate residues in the peptide sequence DEVD. The DEVDsubstrate is labeled with a fluorescent molecule, which exhibits a blueto green shift in fluorescence upon cleavage. Active caspase-3 in theoligonucleotide treated cells is measured by this assay according to themanufacturer's instructions. 48 hours after oligonucleotide treatment,50 uL of assay buffer was added to each well, followed by addition 20 uLof the caspase-3 fluorescent substrate conjugate. Data were obtained intriplicate. Fluorescence in wells was immediately detected(excitation/emission 400/505 nm) using a fluorescent plate reader(SpectraMAX GeminiXS, Molecular Devices, Sunnyvale, Calif.). The platewas covered and incubated at 37° C. for and additional three hours,after which the fluorescence was again measured (excitation/emission400/505 nm). The value at time zero was subtracted from the measurementobtained at 3 hours. The measurement obtained from the untreated controlcells was designated as 100% activity. The data are presented in Table28. Values above or below 100% indicate an increase or decrease incaspase-3 activity, respectively.

Cell proliferation and viability were measured using the CyQuant CellProliferation Assay Kit (Molecular Probes, Eugene, Oreg.) utilizing theCyQuant GR green fluorescent dye which exhibits strong fluorescenceenhancement when bound to cellular nucleic acids. After the 48 houroligonucleotide treatment, the microplate was gently inverted to removethe medium from the wells, which were each washed once with 200 uL ofphosphate-buffered saline. Plates were frozen at −70° C. and thenthawed. A volume of 200 uL of the CyQUANT GR dye/cell-lysis buffer wasadded to each well. The microplate was incubated for 5 minutes at roomtemperature, protected from light. Data were obtained in triplicate.Fluorescence in wells was immediately detected (excitation/emission480/520 nm) using a fluorescent plate reader (SpectraMAX GeminiXS,Molecular Devices, Sunnyvale, Calif.). The measurement obtained from theuntreated control cells was designated as 100% activity. The data arepresented in Table 28. Values above or below 100% indicate an increaseor decrease in caspase-3 activity, respectively.

TABLE 28 Apoptosis and cell viability: comparison of gapmer andgap-disabled oligomeric compounds % cell % caspase- SEQ ISIS # Motifviability 3 activity ID NO 126965 5-10-5 27 2408 39 3306932-1-1-2-1-1-1-1-1-1-1-1-1-3-2 76 119 39 129605 5-10-5 60 156 63 1945632-1-1-2-1-1-1-1-1-1-1-1-1-3-2 51 67 63 180475 5-10-5 30 436 43 3008613-2-1-3-1-3-1-3-3 43 94 43 256899 5-10-5 68 110 60 3041703-2-1-2-1-2-1-2-1-2-3 58 72 60

These data demonstrate that when cells were treated with compounds havethe nucleobase sequence of SEQ ID NOs: 39 and 43, cell viability washigher and caspase-3 activity was lowered in cells treated with thegap-disabled compounds, as compared to cells treated with the gapmercompounds. Comparison of gap-disabled and gapmer compounds having thenucleobase sequence of SEQ ID NOs: 63 and 60 reveals that both cellviability and caspase-3 activity were lowered in the cells treated withthe gap-disabled compounds, as compared to cells treated with the gapmercompounds. These data further illustrate that gap-disabled compounds,like gapmer compounds, are able to modulate cellular pathways.

Example 49 Gap-Disabled vs. Gapmer Oligomeric Compounds: HepatotoxicEffects

A number of chemical modifications have been introduced into oligomericcompounds to increase their usefulness as therapeutic agents and improvetheir pharmacokinetic properties. Of particular interest is theelimination of toxicity caused by oligomeric compounds, which can besignificant in the liver and kidney due to the relatively highaccumulation of oligomeric compounds in these organs. In a furtherembodiment, the hepatotoxic effects of the gapmer compound ISIS 129605(SEQ ID NO: 63; no known target) and the gap-disabled compound ISIS194563 (SEQ ID NO: 63; no known target) were tested in normal mice.Other oligomeric compounds tested included ISIS 118929 (SEQ ID NO: 53),a randomized control ISIS 29848, where N is A, T, C or G, SEQ ID NO:75); and ISIS 148548 (SEQ ID NO: 59), all three of which are gapmeroligomeric compounds with 5-methylcytidines and phosphorothioateinternucleoside linkages throughout.

Normal mice, maintained on a lean diet, were injected with 50 mg/kg ofeach oligomeric compound, twice weekly for 2 weeks. Saline-injectedanimals served as a control group. Each treatment group contained 4animals. Animals were sacrificed at the end of the treatment period.Liver weights were determined at necropsy, and serum was collected foranalysis of liver transaminase levels determined by routine clinicalassays.

The serum transaminases ALT and AST are frequently used as indicators ofhepatotoxicity. ISIS 129605 caused marked increases in both AST and ALTlevels, which were 20 and 17 times, respectively, that observed insaline-treated mice. Conversely, ISIS 194563, which has the samenucleotide sequence as ISIS 129605 but is a gap-disabled compound,caused no increase in ALT and AST levels relative to saline-treatedanimals. Similarly, treatment ISIS 118929, ISIS 148548 or ISIS 29848 didnot result in elevated ALT and AST levels. Increases in liver and spleenweights can also indicate the presence of toxicity. Treatment with ISIS129605 resulted in an increase in liver weight approximately 1.6 timesthat of livers from saline-treated animals. Conversely, treatment withISIS 194563 did not elevate or reduce liver weight. Serum transaminaselevels and liver weight data demonstrate that introduction of 2′-MOEnucleotides into the gap segment of ISIS 129605 reduced the toxicity ofthat compound. Liver weights following treatment with the other gampercompounds were not significantly increased. None of the compoundsresulted in significantly elevated spleen weights.

An additional in vivo experiment was performed, using oligomericcompounds described herein: ISIS 129605 (SEQ ID NO: 63), a gapmer havingthe motif 5-10-5 wherein the wing segments are composed of 2′-MOEnucleotides; ISIS 189525 (SEQ ID NO: 63), a gapmer having the motif5-10-5, wherein the wings are composed of 2′-MOE nucleotides, and alsohaving unmodified cytosines (rather than 5-methylcytosines); ISIS 199041(SEQ ID NO: 63), uniformly composed of 2′-MOE nucleotides; ISIS 199042(SEQ ID NO: 63), a gap-disabled compound having the motif5-2-1-2-1-2-1-1-5; ISIS 199043 (SEQ ID NO: 63), uniformly composed of2′-deoxynucleotides; ISIS 199044 (SEQ ID NO: 63), a 5-10-5 gapmerwherein the wing segments are composed of 2′-O-methyl nucleotides andthe gap is composed of 2′-deoxynucleotides; and ISIS 199046 (SEQ ID NO:63), a 5-10-5 gapmer wherein the wing segments are composed of 2′-MOEnucleotides, and wherein the internucleoside linkages in the wings arephosphodiester and the internucleoside linkages in the gap arephosphorothioate. Also tested were ISIS 199047 (SEQ ID NO: 61) and ISIS199048 (SEQ ID NO: 62), both gapmer compounds with the motif 5-10-5,having wing segments composed of 2′-MOE nucleotides. Unless otherwisenoted, internucleoside linkages are phosphorothioate and cytosines are5-methylcytosines. For each motif presented, emboldened, underlined typeindicates 2′-MOE nucleotides and plain type indicated2′-deoxynucleotides. SEQ ID NOs: 63, 61 and 62 are not perfectlycomplementary to any known target.

Lean mice were treated with 50 mg/kg oligomeric compound, twice weeklyfor 3 weeks. The serum transaminases ALT and AST, indicators oftoxicity, were measured by routine clinical analysis at the end of thestudy. ISIS 129605 treatment resulted in AST and ALT levelsapproximately 6 and 5 times those of saline-treated mice, respectively.ISIS 199044 resulted in dramatically elevated AST and ALT, approximately15 and 9 times those of saline-treated mice, respectively. Treatmentwith ISIS 199048 also resulted in elevated AST and ALT, approximately 10and 15 times those of saline-treated mice, respectively. Thegap-disabled compound ISIS 199042 did not significantly elevate ALT andAST levels, demonstrating that an additional gap-disabled compoundexhibits significantly fewer toxic properties than the gapmer versionhaving the same nucleotide sequence. ISIS 189525, ISIS 199041, ISIS199043, ISIS 199046 and ISIS 199047 similarly did not causesignificantly elevated ALT and AST levels, illustrating that variouschemical modifications of SEQ ID NO: 63 exhibit fewer toxic propertiesrelative to ISIS 129605.

Liver weights, increases in which can also indicate toxicity, were alsomeasured at the end of the study. In accordance with the observationthat ISIS 199042 did not elevate ALT and AST levels, this compound didnot significantly change liver weight. ISIS 199041 and ISIS 199046 didnot cause increases in liver weight. However, ISIS 129605 and ISIS199044, which did exhibit toxic properties as judged by ALT and ASTlevels, increased liver weight by approximately 1.6 and 1.8 times thatof liver weights from saline-treated mice. These data furtherdemonstrate the toxic properties of these compounds. Although ISIS189525 and ISIS 199043 did not elevate ALT and AST levels, treatmentwith these compounds resulted in approximately 1.4-fold increases inliver weights relative to livers from saline-treated mice.

These in vivo studies illustrate that chimeric oligomeric compoundshaving at least 9 alternating 3′-endo and 2′-endo regions amelioratehepatotoxicity, thereby improving the pharmacokinetic properties of thecompounds. Thus, these compounds have applications in the development oftherapeutic agents.

Example 50 In Vivo Comparison of Gapmer and Gap-Disabled OligomericCompounds Targeted to JNK1: Target Reduction and Toxicity

In a further embodiment, gap-disabled and gapmer oligomeric compoundstargeted to both human and mouse jun N-terminal kinase-1 (JNK1) weretested for their effects on both toxicity and target reduction in vivo.The gap-disabled compound ISIS 345888 (SEQ ID NO: 37) and the gapmercompound ISIS 307754 (SEQ ID NO: 37) are both shown in Table 13 and wereselected for this study.

Male Balb/c mice, 6 to 7 weeks of age, received twice weeklyintraperitoneal injections of 12.5, 25 or 50 mg/kg of either ISIS 307754or ISIS 345888, for a period of three weeks. ISIS 141923 (SEQ ID NO: 64)was used as a negative control oligomeric compound and was injected at50 mg/kg. Saline-injected animals served as a control group and wereinjected in the same manner as the oligomeric compounds. Each treatmentgroup contained 4 animals. Body weights were monitored throughout thestudy (Days 1, 5, 8, 12, 15 and 19). Two days following the finalinjection, animals were sacrificed (Day 20). Liver and spleen weights,increases in which can indicate toxicity, were determined at time ofnecropsy. Serum was collected for analysis of the liver transaminaseALT, an indicator of toxicity. ALT levels were determined by routineclinical analysis. Liver tissue was collected for measurement of targetmRNA expression by real-time PCR. Liver and kidney tissue were evaluatedfor concentration of total and full-length oligomeric compound bycapillary gel electrophoresis.

ALT levels are shown in Table 29, in international units per liter(IU/L), with the saline control levels included for comparison. Body,liver and spleen weights are also presented in Table 29. Body weightsare shown as percentage relative to the weight of each animal at thestart of the study. Liver and spleen weights are normalized tosaline-treated control weights.

TABLE 29 Indicators of toxicity: gap-disabled vs. gapmer oligomericcompounds targeted to JNK1 Liver Spleen ALT Body Weights Weight WeightIU/L % relative to Day 1 % relative to saline Dose, Day Day Day Day DayDay Day Day Treatment mg/kg 20 5 8 12 15 19 20 20 Saline none 46 100 106104 108 108 100 100 141923 50 64 100 104 105 107 109 99 106 307754 50292 101 102 103 102 105 131 147 307754 25 218 98 101 101 105 107 118 122307754 12.5 40 101 105 106 106 109 109 120 345888 50 58 100 104 103 106106 101 125 345888 25 103 102 105 105 107 110 110 124 345888 12.5 48 98103 103 107 110 107 119

From these data, it is evident that at doses of 25 or 50 mg/kg,treatment with the gap-disabled compound ISIS 345888 resulted inmarkedly lower ALT levels, relative to treatment with the gapmercompound ISIS 307754. These data further reveal that at the 25 and 50mg/kg doses, ISIS 307754 caused increases in liver weight, relative tosaline-treatment. The 12.5 mg/kg dose of ISIS 307754 did not increaseliver weight, relative to saline-treatment. None of the doses of thegap-disabled compound ISIS 345888 resulted in an increase in liverweight, relative to saline-treatment. Thus, ISIS 345888 exhibits fewertoxic properties than ISIS 307754.

Oligomeric compounds isolated from kidney and liver tissue weresubjected to capillary gel electrophoresis, to determine theconcentrations of total and full-length oligomeric compound. The totalconcentration of oligomeric compound following treatment with ISIS307754 (gapmer) was 163 μg/g in kidney and 176 μg/g in liver.Full-length ISIS 307754 represented 94% of the total compound present inkidney and 98% of the total compound present in liver. The totalconcentration following treatment with ISIS 345888 was 126 μg/g inkidney and 174 μg/g in liver. Full-length ISIS 345888 represented 82% ofthe total compound present in kidney and 78% of the total compoundpresent in liver. These data demonstrate that full-length ISIS 345888accumulates in liver and kidney tissue.

Liver RNA was analyzed for JNK1 expression levels by quantitativereal-time PCR as described by other examples herein, using thehousekeeping gene cyclophilin A to normalize RNA levels among samples.In Table 30, JNK1 mRNA expression levels are shown as normalized tosaline-treated control JNK1 levels.

TABLE 30 Target reduction and serum transaminases: gap-disabled vs.gapmer oligomeric compounds targeted to JNK1 JNK1 Dose, mRNA % Treatmentmg/kg control 141923 50 93 307754 50 15 307754 25 16 307754 12.5 31345888 50 23 345888 25 26 345888 12.5 49

These results demonstrate a substantial reduction in target expressionfollowing treatment with both the gap-disabled and gapmer compounds.Furthermore, the hepatoxicity caused by the gapmer, as judged by liverweights and ALT levels, is ameliorated by the introduction of 2′-MOEnucleotides into the gap segment. The significant reduction of JNK1 mRNAin livers of mice treated with ISIS 345888 also illustrates that theconcentration of ISIS 345888 accumulated in the liver is an amountsufficient to elicit substantial target reduction.

Example 51 Antisense Inhibition by Gap-Disabled Oligomeric CompoundsTarget to Human C-Raf

In a further embodiment, a series of oligomeric compounds was designedto target human C-raf RNA, using publicly available sequences (GenBankaccession number X03484.1, incorporated herein as SEQ ID NO: 29; and asequence assembled from GenBank accession numbers AC026153.10 andAC018500.2, incorporated herein as SEQ ID NO: 30). The compounds areshown in Table 31. “Target site” indicates the first (5′-most)nucleotide number on the particular target sequence to which thecompound binds. All compounds in Table 31 are chimeric oligomericcompounds, comprising regions that alternate between 3′-endo regions and2′-endo regions, also known as gap-disabled compounds. The motif of eachcompound in Table 31 is 3-2-1-2-1-2-1-2-1-2-3, where the numberindicates the number of nucleosides in that region. Regions consistingof 2′-MOE nucleotides are indicated by bold, underlined type and theremaining regions in plain type consist of 2′-deoxynucleotides. Allinternucleoside linkages are phosphorothioate linkages, and allcytosines are 5-methylcytosines.

The compounds were tested for their effect on human C-raf mRNA levels byquantitative real-time PCR as described in other examples herein. Dataare averages from two experiments in which A549 cells were treated with75 nM of the compounds in Table 31. ISIS 18078 (SEQ ID NO: 8), whichdoes not target Raf kinase C, was used as a negative controloligonucleotide in this assay.

TABLE 31 Antisense inhibition by gap-disabledoligomeric compounds targeted to human C-raf Target Seq SEQ Target % IDIsis # Region ID NO Site Sequence 5′ to 3′ Inhib NO 336818 Coding 29 94attcttaaacctgagggagc 26 76 336819 Coding 29 144 tgatcgtcttccaagctccc 4677 336820 Coding 29 198 gagagatgcagctggagcca 61 78 336821 Coding 29 249tgccatcatctgatgcccgg 77 79 336822 Coding 29 268 ttagaaggatctgtgagttt 6780 336823 Coding 29 327 gcacattgaccactgttctt 43 81 336824 Coding 29 367agtgctttcataaggcagtc 38 82 336825 Coding 29 392 ctctggttgcaggcccctca 5983 336826 Coding 29 413 aagtctgaacactgcacagc 57 84 336827 Coding 29 433ttacctttgtgttcgtggag 75 85 336828 Coding 29 466 gcagcatcagtattccaatc 5886 336829 Coding 29 543 tcttccgagcaaagttgtgt 35 87 336830 Coding 29 591tgagcaggaatttctgacag 53 88 336831 Coding 29 701 tggaaacaataagagttgtc 4089 336832 Coding 29 776 ggaaacagactctcgcatac 38 90 336833 Coding 29 800gtgctgagaactaacaggca 78 91 336834 Coding 29 909 tgaccatgtggacattaggt 7092 336835 Coding 29 931 ctgtccacaggcagcgtggt 40 93 336837 Coding 29 954gaaggtgaggctgattcgct 43 94 336838 Coding 29 982 cacgaggcctaattttgttt 9195 336839 Coding 29 1110 atttcccaataatagcttga 68 96 336840 Coding 291141 gcaacatctccgtgccattt 48 97 336841 Coding 29 1228tcctgaaggcctggaattgc 53 98 336842 Coding 29 1284 ccgtgttttgcgcagaacag 5099 336843 Coding 29 1313 caggttgtcctttgtcatgt 17 100 336844 Coding 291361 ggacatgcaggtgtttgtag 27 101 336845 Coding 29 1416attagctggaacatctgaaa 19 102 336846 Coding 29 1447 atgcaaatagtccattccct21 103 336847 Coding 29 1490 gaaatatattgttggatttc 61 104 336848 Coding29 1536 cgtgactttactgttgccaa 34 105 336849 Coding 29 1594gggccatccagaggacagag 58 106 336850 Coding 29 1650 tgaagatgatctgatctcgg31 107 336851 Coding 29 1788 atatagcttactaagatctg 33 108 336852 Coding29 1832 aatggaagacaggatctggg 34 109 336853 Coding 29 1928cttcggtagagagtgttgga 52 110 336854 Coding 29 1955 tatcctcagtgtgggctgcc39 111 336855 Coding 29 2010 tgcaaagtcaactagaagac 49 112 336856 Coding29 2068 ttctgcctctggagaaaggg 20 113 336857 Intron 29 2144aggtccttagcagagcttct 33 114 336858 Intron 29 2177 aaatggcttccttctcccag13 115 336859 Intron 29 2255 tacagaaggctgggccttga 67 116 336860 Intron29 2317 tttttgtactaccatcaaca 50 117 336861 Intron 29 2351acttcctctaaatactcatg 24 118 336862 Intron 29 2399 tccacatcagggctggactg32 119 336863 Intron 29 2430 gaagctgatttccaaaatcc 14 120 336864 Intron29 2458 tcccgcctgtgacatgcatt 39 121 336865 Intron 29 2484accactctctgaagaaagtc 25 122 336866 Intron 29 2502 gtgccttatgtgcaaaatgt36 123 336867 Intron 29 2532 ggcggccagagtctcggcag 13 124 336868 Intron29 2566 ctaagaaaagttccatagta 14 125 336869 Intron 29 2604gaagctgtgaaaggaggacg 9 126 336870 Intron 29 2630 gggcagctcctggaagacaa 31127 336871 Intron 29 2746 tgtatacacatgatgtgact 25 128 336872 Intron 302834 aacatagctatttgaagcta 47 129 336873 Intron 30 27366aagcaataatttcaatttct 35 130 336874 Intron 30 27473 gcccagcttaacgtgtattt12 131 336875 Intron 30 27513 tcatcaggcccagcttaacg 52 132 336876 Intron30 27520 ccatccatggaaacattatc 35 133 336877 Intron 30 28081acagcatctaacatcactgt 24 134 336878 Intron 30 28103 agtcaatctcccgaggatag38 135 336879 Intron 30 28215 agtgacgctttccaagaaga 27 136 336880 Intron30 28503 atgtaagctaacgatgaata 10 137 336881 Exon-Exon Junction 30 28528ttccctgggctattctccca 51 138 336882 Exon-Exon Junction 30 28577aattgagaattacactcacc 55 139 336883 Exon-Exon Junction 30 28613aacgcctcctaaattgagaa 27 140 336884 Exon-Exon Junction 30 28624tggattggcttagggaccca 25 141 336885 Exon-Exon Junction 30 28700actattttgcccttatgaag 81 142 336886 Exon-Exon Junction 30 28886tcttaaaatctactctgaaa 29 143 336887 Exon-Exon Junction 30 29191cttaactgtcttaaaatcta 47 144 336888 Exon-Exon Junction 30 29199tgaaaaatgtacttttctat 51 145 336889 Exon-Exon Junction 30 29273aaagttttctttaaacaatg 44 146 336890 Exon-Exon Junction 30 29462gcccatgttctcagaataaa 63 147 336891 Exon-Exon Junction 30 29641aatctaggtctgttgaactc 6 148 336892 Exon-Exon Junction 30 29665aaggtaatttgctcaaggcc 46 149 336893 Exon-Exon Junction 30 29713agaaaactgggactctaaga 60 150 336894 Exon-Exon Junction 30 29732tatttctatctgaaaaataa 48 151 336895 Exon-Exon Junction 30 29751aacaaacctatgaagtaggt 59 152 337561 Exon 1: Intron 1 30 29773tgccacctacctgagggagc 43 153 337562 Exon 10: Intron 10 30 20510attcttaaacctggtaagaa 64 154 337563 Exon 11: Intron 11 30 20743gttcacataccactgttctt 48 155 337564 Exon 12: Intron 12 30 27195gcacattgacctacaaacaa 57 156 337565 Exon 13: Intron 13 30 27308gagctcttaccctttgtgtt 45 157 337566 Exon 14: Intron 14 30 30025tgcaacttacaaagttgtgt 67 158 337567 Exon 15: Intron 15 30 30334tcttccgagcctacaacaag 43 159 337568 Exon 2: Intron 2 30 30492aatgccttacaagagttgtc 48 160 337569 Exon 3: Intron 3 30 34981gtgctgagaactaggaggag 63 161 337570 Exon 4: Intron 4 30 35135gccctattacctcaatcatc 48 162 337571 Exon 5: Intron 5 30 38855gaattgcatcctgaaacaga 69 163 337572 Exon 7: Intron 7 30 38883ggaaaagtacctgattcgct 43 164 337573 Exon 8: Intron 8 30 38991gaaggtgaggcttaatagac 84 165 337574 Intron 1: Exon 2 30 39462cacgaggcctctgaaacaag 60 166 337575 Intron 10: Exon 11 30 39580ccaagcttaccgtgccattt 65 167 337576 Intron 12: Exon 13 30 47482gcaacatctcctgcaaaatt 40 168 337577 Intron 13: Exon 14 30 47567ttctactcaccgcagaacag 28 169 337578 Intron 15: Exon 16 30 48476tctactcactccattccctg 38 170 337579 Intron 16: Exon 17 30 51633atgcaaatagctgtgaaggg 58 171 337580 Intron 2: Exon 3 30 51680caaaggatactgttggattt 76 172 337581 Intron 4: Exon 5 30 53471agaaatatatctcaatgctt 58 173 337582 Intron 6: Exon 7 30 53590agattctcaccatccagagg 79 174 337583 Intron 7: Exon 8 30 54149acagacttacctgatctcgg 44 175 337584 Intron 8: Exon 9 30 54289tgaagatgatctaagggaaa 65 176 337585 Intron 9: Exon 10  30 54615ggaagacaggatctgaaaca 56 177

These data reveal that SEQ ID NOs 77, 78, 79, 80, 81, 83, 84, 85, 86,88, 91, 92, 94, 95, 96, 97, 98, 99, 104, 106, 110, 112, 116, 117, 129,132, 138, 139, 142, 144, 145, 146, 147, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 171,172, 173, 174, 175, 176 and 177 exhibited at least 43% inhibition ofhuman C-raf mRNA expression in this assay.

Example 52 Antisense Inhibition by Gap-Disabled Oligomeric CompoundsTarget to Mouse SRC-2

In a further embodiment, a series of oligomeric compounds was designedto target mouse SRC-2 RNA, using publicly available sequences (GenBankaccession number U39060.1, incorporated herein as SEQ ID NO: 31; thecomplement of nucleotides 10220000 to 10460000 of the sequence withGenBank accession number NW_(—)000149.1, incorporated herein as SEQ IDNO: 32; and GenBank accession number AK028964.1, incorporated herein asSEQ ID NO: 33). The compounds are shown in Table 32. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the compound binds. All compounds in Table 32 arechimeric oligomeric compounds, comprising alternating 3′-endo regionsand 2′-endo regions, also known as gap disabled compounds. The motif ofeach compound in Table 19 is 3-2-1-2-1-2-1-2-1-2-3, where the numberindicates the number of nucleosides in that region. Regions consistingof 2′-MOE nucleotides are indicated by bold, underlined type and theremaining regions in plain type consist of 2′-deoxynucleotides. Allinternucleoside linkages are phosphorothioate linkages, and allcytosines are 5-methylcytosines.

The compounds were tested for their effect on mouse SRC-2 mRNA levels byquantitative real-time PCR as described in other examples herein. Dataare averages from two experiments in which b.END cells were treated with50 nM of the compounds in Table 32. ISIS 337599 (GTGCGCGCGAGCCCGAAATC,SEQ ID NO: 178), which does not target Raf kinase C, is a gap-disabledcompound having the same motif as the compounds in Table 32 and was usedas a negative control compound in this assay.

TABLE 32 Antisense inhibition by gap-disabled oligomericcompounds targeted to human Raf kinase C TARGET SEQ SEQ ID TARGET % IDISIS # Region NO SITE SEQUENCE (5′ to 3′) INHIB NO 337600 5′ UTR 31 174tatcagcaactgtgcctgta 6 179 337601 5′ UTR 31 193 cccactcatcttgaacacat 21180 337602 Coding 31 479 tctgcacttcatctatgttg 45 181 337603 Coding 31646 cagctcttcttggttatacc 37 182 337604 Coding 31 1170tgtctcagaacttcatggtg 34 183 337605 Coding 31 1257 gaacggatgagtttgctctt 9184 337606 Coding 31 1272 tcattagtagtctgagaacg 0 185 337607 Coding 311426 acctgggttcccactgcaca 49 186 337608 Coding 31 1462gggaaaatttatattgctac 9 187 337609 Coding 31 1491 atgcccatttgttcctttgg 22188 337610 Coding 31 2244 tgcttctccttgagcgaggt 31 189 337611 Coding 312509 aggatctgtcttactgtcca 53 190 337612 Coding 31 2519tgttactggcaggatctgtc 20 191 337613 Coding 31 2625 tgcaaatcatccaaaatctc26 192 337614 Coding 31 2700 atggcttgcttgtcaactga 53 193 337615 Coding31 2705 tgatgatggcttgcttgtca 35 194 337616 Coding 31 2720gttgcatgaggtcattgatg 31 195 337617 Coding 31 2804 gtgggttattaaaagtgctc 7196 337618 Coding 31 2809 tggtcgtgggttattaaaag 16 197 337619 Coding 312819 ccagttgccctggtcgtggg  39 198 337620 Coding 31 2824cctgcccagttgccctggtc 10 199 337621 Coding 31 2839 ctggtttggcaataacctgc 1200 337622 Coding 31 2885 gtccagcaccagttgggctt 27 201 337623 Coding 312890 gaaaggtccagcaccagttg 33 202 337624 Coding 31 2900tgattggtgggaaaggtcca 11 203 337625 Coding 31 2910 ctactgtttctgattggtgg33 204 337626 Coding 31 2934 ggctgaggtatcactgagta 62 205 337627 Coding31 2939 ttcctggctgaggtatcact 34 206 337628 Coding 31 2949ttacccatcattcctggctg 36 207 337629 Coding 31 3182 gtctttggccaggctggctg27 208 337630 Coding 31 3513 tggctctggctgaccagttc 20 209 337631 Coding31 3650 agtttggatcttgcatggga 28 210 337632 Coding 31 3789ttctgctgtgcttggaggcg 24 211 337633 Coding 31 4137 gtcgtagccccagtaaagcc 2212 337634 3′ UTR 31 4833 agttgcactacggtgaatgc 43 213 337635 Exon 1a: 3277182 gcatctttaccacttcagga 39 214 Intron 1c 337636 Exon 11: 32 200699gaaaactcacctggtcactg 0 215 Intron 11 337637 3′ UTR 33 2520aacaggtcgagctcagtagt 34 216

These data demonstrate that SEQ ID NOs 180, 181, 182, 183, 186, 188,189, 190, 191, 192, 193, 194, 195, 198, 201, 202, 204, 205, 206, 207,208, 209, 210, 211, 213, 214 and 216 demonstrated at least 20%inhibition of mouse SRC-2 in this assay. These results provide anotherexample of target inhibition by gap-disabled oligomeric compounds.

Example 53 Recombinant Human RNase H Analysis

RNase H is a cellular endonuclease which cleaves the RNA strand of anRNA:DNA duplex. It is known in the art that single-stranded antisenseoligomeric compounds which are “DNA-like” or have DNA-like regionselicit RNase H activity. Activation of RNase H, therefore, results incleavage of the RNA target, thereby allowing oligonucleotide-mediatedinhibition of gene expression.

In a further embodiment, the ability of oligomeric compounds to elicitRNase H activity was tested using RNase H activity assays. Where themotif of each compound is indicated, 2′-MOE nucleotides are in bold,underlined type and 2′-deoxynucleotide regions are in plain type. Thenumber in each region represents the number of nucleotides in thatregion. Oligomeric compounds tested included ISIS 300861 (SEQ ID NO:43), a gap-disabled compound having the motif 3-2-1-3-1-3-1-3-3 andphosphorothioate (P═S) internucleoside linkages throughout the compound,and ISIS 335114 (SEQ ID NO: 43), also a gap-disabled compound having themotif 3-2-1-3-1-3-1-3-3 and phosphodiester (P=0) internucleosidelinkages throughout the compound. Also tested was ISIS 335112 (SEQ IDNO: 43), a chimeric oligonucleotide 20 nucleotides in length, having a10-nucleotide gap segment flanked on both sides (5′ and 3′) by5-nucleotide wing segments, wherein the gap segment consists of2′-deoxynucleotides and the wing segments consist of 2′-MOE nucleotides.Internucleoside linkages are phosphodiester (P═O) throughout thecompound. An additional oligomeric compound tested was ISIS 335033 (SEQID NO: 43), uniformly composed of 2′-deoxynucleotides withphosphodiester (P═O) internucleoside linkages throughout the compound.In these compounds, all cytosines are 5-methylcytosines.

RNase H1 activity was evaluated using 40 oligoribonucleotides of mouseglucagon receptor RNA (GTTGGAGGCAATGGCAAGAAGGCAAAGCTCTTCAGGAGGA,incorporated herein as SEQ ID NO: 217) as the target RNA. This targetRNA was radiolabelled with ³²P at the 5′-end as described by Wu et al.(J. Biol. Chem., 2001, 276, 23547-23553). In a volume of 100 μA, 100 nMof radiolabelled RNA and 200 nM of oligomeric compound were incubated ina reaction containing 20 mM Tris HCl, pH 7.5, 20 mM KCl, 1 mM MgCL₂, 0.1mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 4% RNaseOUT™(Invitrogen Corporation, Carlsbad, Calif.). Reactions were melted byheating to 95° C. for 5 minutes then allowed to cool slowly to roomtemperature. Formation of the heteroduplex between the target RNA and anoligomeric compound was confirmed by the shift in mobility between thesingle-stranded end labeled sense RNA and the annealed duplex onnon-denaturing polyacrylamide gels. The resulting heteroduplexes weretested as substrates for digestion by recominant human RNase H1, whichwas expressed and purified as described by Wu et al. (J. Biol. Chem.,2004, 279, 17181-17189). An aliquot of annealed heteroduplex reactionwas removed for use as the t=0 timepoint. 70 ng of purified recombinantRNase H1 in a solution of 50 mM Tris HCl, pH 7.5, 50 mM NaCl, 50%glycerol, 1 mM TCEP and 4% RNaseOUT™ was added to 100 μL of the duplexreaction and was incubated at 37° C. The reaction was terminated at 15,60 and 240 minute timepoints by the addition of 4M Urea and 20 mM EDTA.The reactions were heated at 90° C. for 2 minutes and the reactionproducts were resolved on a 12% polyacrylamide gel containing 7 M Ureaand visualized and quantitated using a PhosphorImager™ and IMAGEQUANT™Software (Molecular Dynamics, Sunnyvale, Calif.).

Recombinant human RNase H1 was tested for its ability to cleave fourdifferent heteroduplexes formed between the target RNA and each of theoligomeric compounds ISIS 335112, ISIS 335033, ISIS 335114 and ISIS300861. The percentage of target RNA cleaved was calculated using thefollowing formula: [(fraction of RNA cleaved/total RNA input)×100]−%background. The data from the 15, 60 and 240 minute time points werenormalized to the data from the t=0 timepoint. The results are shown inTable 33.

TABLE 33 Recombinant human Rnase-H1 mediated cleavage of heteroduplexes% target mRNA cleavage Reaction ISIS 335112 ISIS 335033 ISIS 335114 ISIS300861 time Gapmer 2′-deoxy Gap-disabled Gap-disabled (minutes) P = O P= O P = O P = S 15 3 5 1 0 60 15 30 1 1 240 38 74 1 1

These data demonstrate that whereas ISIS 335112 (gapmer) and ISIS 335033(uniform 2′-deoxy) oligomeric compounds elicited RNase H1-mediatedcleavage, the gap-disabled compounds (ISIS 335114 or ISIS 300861) wereunable to utilize recombinant RNase H1 to effect the detectable cleavageof target mRNA in this assay.

Example 54 Immunoprecipitated RNase H Activity

In a further embodiment, the ability of gap-disabled oligomericcompounds to utilize immunoprecipitated RNase H1 and RNase H2 to directthe cleavage of target RNA was tested. Polyclonal antibodies weregenerated by Biosolutions (Ramona, Calif.) using RNase H1 and RNase H2proteins purified as described by Wu, et al. (J. Biol. Chem., 2004, 279,17181-17189). Immunoprecipitations were also performed as described byWu, et al. (J. Biol. Chem., 2004, 279, 17181-17189). Duplex formationwas performed as described herein, using ISIS 300861 (SEQ ID NO: 43,gap-disabled), ISIS 335112 (SEQ ID NO: 43, gapmer) and ISIS 335033 (SEQID NO: 43, uniform 2′-deoxy) as the oligomeric compounds and the mouseglucagon receptor (SEQ ID NO: 217) as the target RNA. The cleavage assaywas performed as described herein for recombinant RNase H1, using RNaseH1 or RNase H2 immunoprecipitated with 10 μg of the respective antibodyper 1 mg total cellular protein. Samples of the cleavage assay werecollected at t=0 (start of the reaction), 15, 60 and 180 minutes andproducts were resolved by denaturing polyacrylamide electrophoresis andvisualized by using a PhosphorImager™ and IMAGEQUANT™ Software(Molecular Dynamics, Sunnyvale, Calif.). Whereas both ISIS 335033(uniform 2′ deoxy) and ISIS 335112 (gapmer) were able to direct cleavageof the target RNA by both immunoprecipitated RNase H1 and RNase H2, ISIS300861 (gap-disabled) did not elicit detectable cleavage of the targetRNA by immunoprecipitated RNase H1 or RNase H2 in this assay.

Example 55 In Vitro Nuclease Assay

In a further embodiment, the ability of gap-disabled compounds to elicittarget cleavage in subcellular fractions was evaluated. HeLa cellnuclear, nuclear membrane and cytosolic fractions were isolated asdescribed previously (Dignam, et al., Nucleic Acids Research., 1983, 11,1475-1489) and used to test the ability of gap-disabled oligomericcompounds to elicit target reduction. Following isolation of thesubcellular fractions, the cleavage assay was performed as described forrecombinant RNase H1.

Duplexes between the mouse glucagon receptor RNA and oligomeric compoundwere prepared as described herein, using as the oligomeric compoundsISIS 335033 (SEQ ID NO: 43, uniform 2′-deoxy), ISIS 335112 (SEQ ID NO:43, gapmer), ISIS 300861 (SEQ ID NO: 43, gap-disabled) and ISIS 298683(SEQ ID NO: 43, uniform 2′-MOE). Annealed duplexes (10 μl) wereincubated with 3 μg of the HeLa cytosolic extract at 37° C. The assaywas also performed with a 4-fold higher concentration of cytosolicextract. Samples were collected at t=0 (reaction start time), 15, 60 and180 minutes. The reaction was terminated by phenol/chloroform extractionand ethanol precipitated with the addition of 10 μg of tRNA as acarrier. Pellets were resuspended in 10 μl of denaturing loading dye andproducts were resolved on 12% denaturing acrylamide gels as describedherein. Visualization of cleavage patterns by PhosphorImager™ detectionrevealed that while HeLa cytosolic extracts were capable of supportingtarget cleavage mediated by ISIS 335112 (gapmer) and ISIS 335033(uniform 2′-deoxy) in a time-dependent manner, this fraction was unableto support target reduction by ISIS 300861 (gap-disabled). Additionally,ISIS 298683 (uniform 2′-MOE) was unable to direct cleavage in thecytosolic extracts.

The ability of ISIS 300861 (gap-disabled) and ISIS 335112 (gapmer) tomediate target cleavage was also tested in the nuclear fraction isolatedfrom HeLa cells. In this assay, the mouse glucagon receptor target RNAcontained a 3′ phosphorothioate cap, to improve its resistance toexonuclease activity, which, as is known in the art, is present innuclear extracts and results in non-specific degradation of the targetRNA. Annealed duplexes (10 μl) were incubated with 3 μg of the HeLanuclear extract at 37° C. The assay was performed both in the presenceand absence of beta-mercapoethanol. Samples were collected at t=0 (startof the reaction), 10 and 60 minutes. Resolution of the products on adenaturing polyacrylamide gel, followed by PhosphorImager™ detection,revealed that ISIS 300861 (gap-disabled) and ISIS 355112 (gapmer)elicited target cleavage in HeLa nuclear extracts in a time-dependentmanner, both in the presence and absence of beta-mercaptoethanol. Afour-fold higher concentration of nuclear extract was also able tosupport cleavage by both compounds.

The assay was also performed using HeLa nuclear membrane extract assource of RNase activity. Annealed duplexes (10 μl) were incubated with3 μg of the HeLa nuclear membrane extract at 37° C. Neither ISIS 331112(gapmer) nor ISIS 335114 (gap-disabled) was able to elicit cleavage ofthe target RNA in HeLa nuclear membrane extracts.

Together, these data reveal that the enzyme activity responsible for thecleavage of duplexes formed between gap-disabled oligomeric compoundsand target RNAs resides in the nuclear fraction of the cell, not in thecytosolic or nuclear membrane fractions.

In the nuclear extracts, comparison of the target RNA cleavage patternto a molecular weight ladder revealed that cleavage of the target RNAoccurred only at nucleobase positions complementary to a 2′deoxynucleotide of the gap-disabled oligomeric compound, i.e. thecleavage sites were positioned within the 2′-deoxynucleotide gaps.Furthermore, within the target site for ISIS 300861, cleavage occurredpreferentially at guanines

Example 56 Influence of Divalent Cations on RNase Activity inSubcellular Extracts

Multiple RNase H-like activities exist in human cells, and theseactivities are differentially activated by magnesium and manganese (Wuet al., J. Biol. Chem., 2004, 279, 17181-17189). Thus, it was ofinterest to determine the influence of these divalent cations on theability of RNase enzymes to cleave heteroduplexes comprisinggap-disabled oligomeric compounds.

ISIS 300861 (SEQ ID NO: 43, gap-disabled) and ISIS 335112 (SEQ ID NO:43, gapmer) were tested for their ability to direct RNase mediatedcleavage in the presence of manganese or magnesium. Duplex formation andsubcellular fractionation of HeLa cells were performed as describedherein. The cleavage assay was also conducted as described herein, withthe addition of 0.05 mM magnesium, 5 mM magnesium, 0.05 mM manganese or5 mM manganese. The cleavage reaction was terminated at t=0 (start ofthe reaction), 15, 60 and 120 minutes, and samples from each of thesetimepoints were resolved on a denaturing polyacrylamide gel. Cleavageproducts were detected using a PhosphorImager™.

In nuclear extracts, prepared as described herein, both magnesium- andmanganese-dependent degradation of ISIS 300861 (gap-disabled) and ISIS335112 (gapmer) heteroduplexes was observed. The cleavage activity inthe presence of 0.05 mM manganese was approximately equal to thatobserved in the presence of 5 mM magnesium, demonstrating that manganeseis more effective than magnesium at enhancing RNase activity in HeLacell nuclear extracts.

The influence of divalent cations on cleavage activity was similarlytested in cytosolic extracts. The assay was performed as describedherein. In the presence of either 5 mM magnesium or 5 mM manganese, ISIS300861 (gap-disabled) did not elicit cleavage of the target RNA. ISIS335112 (gapmer) was, however, able to direct cleavage of the target RNAin cytosolic extracts.

The effects of divalent cations on cleavage by immunoprecipitated RNaseH1 were also evaluated. Duplex formation between the target RNA and ISIS300861(gap-disabled) or ISIS 335112 (gapmer) was conducted as describedherein. Immunoprecipitation and the cleavage assay were performed asdescribed herein, with the addition of 0.05 mM magnesium, 5 mMmagnesium, 0.05 mM manganese or 5 mM manganese to the cleavage assay.ISIS 335112 (gapmer) resulted in target RNA cleavage in the presence ofeither divalent cation at all concentrations. In contrast to the resultsobserved in the absence of divalent cation, the addition of 5 mMmanganese allowed the gap-disabled compound ISIS 300861 to direct thecleavage of the target RNA by immunoprecipitated RNase H1 in a patternconsistent with that observed for gap-disabled cleavage activity innuclear extracts. Coupled with the observation that without additionalmanganese, a gap-disabled compound was unable to utilizeimmunoprecipitated RNase H1 to effect target RNA cleavage, these datademonstrate that additional manganese is required for immunoprecipitatedRNase H1 to cleave heteroduplexes formed between a gap-disabledoligomeric compound and a target RNA.

A similar assay was performed using immunoprecipitated RNase H2,however, neither the gap-disabled nor gapmer oligomeric compoundelicited target RNA cleavage by immunoprecipitated RNase H2, regardlessof the presence of a divalent cation in the cleavage assay.

Further tested was the effect of divalent cations on the activity ofrecombinant human RNase H1. The assay was performed as described herein,using ISIS 300861 as the gap-disabled compound and ISIS 335112 as thegapmer compound. Manganese at a concentration of 5 mM was added to thecleavage assay. In contrast to the results described for the activity ofrecombinant RNase H1 in the absence of additional manganese, in thepresence of 5 mM manganese ISIS 300861 was able to direct cleavage ofits target RNA by recombinant RNase H1. The cleavage pattern mimickedthose observed for nuclear extracts and for immunoprecipitated RNase H1in the presence of manganese. These data demonstrate that manganese isrequired for recombinant RNase H1 to cleave heteroduplexes formedbetween a gap-disabled oligomeric compound and a target RNA.

To extend the observation that the presence of manganese influences thepotency of gap-disabled oligomeric compounds, the extent of cleavage andthe rate at which it occurs were evaluated as a function of manganeseconcentration. Duplex formation between ISIS 300861 and the mouseglucagon target RNA was performed as described herein. A cleavage assaywas performed as described herein using recombinant human RNase H1, withthe addition of manganese at 0.5, 1, 5, 20 or 50 mM. To measure the rateat which cleavage occurs, reactions were terminated at t=0 (start of thereaction), 10, 60 and 180 minutes and the percentage of RNA cleaved wascalculated as described herein, using the t=0 timepoint to normalize thedata from the 10, 60 and 180 minute timepoints. The data are shown inTable 34.

TABLE 34 Dependence of RNA cleavage by recombinant RNase H1 on manganeseconcentration Time Concentration of manganese (mM) (minutes) 0.5 1 5 2050 10 3 56 68 63 0 60 6 69 74 69 0 180 12 75 75 70 12

These data demonstrate concentration-dependent cleavage at 0.5 and 1 mMmanganese, however, the addition of 5 or 20 nM manganese did not furtherincrease target cleavage. The addition of 50 mM manganese inhibitedcleavage of the target RNA.

A comparison of cleavage rates achieved by ISIS 335112 (gapmer) and ISIS300861 (gap-disabled) was conducted. Duplexes formed with the target RNAand ISIS 335112 were cleaved at rates of 0.7 and 1.3 nM per minute inthe presence of 50 and 500 uM manganese, respectively. Duplexes formedwith the target RNA and ISIS 300861 were cleaved at 0.1 and 0.3 uM perminute at manganese concentrations of 50 and 500 uM, respectively. Thesedata demonstrate that cleavage elicited by the gapmer oligomericcompound occurs at a higher rate than that elicited by the gap-disabledoligomeric compound.

Example 57

siRNA-Mediated Disruption of RNase H1 Activity: Influence onGap-Disabled Oligomeric Compound Potency

In a further embodiment, the participation of RNase H1 in the cleavageof target RNA mediated by gap-disabled oligomeric compounds was testedfollowing disruption of cellular RNase H1 mRNA by siRNAs. Because siRNAselicit target reduction through mechanisms not dependent on RNase H1,the use of siRNAs to disrupt the expression of RNase H1 is a method bywhich the activity of RNase H1 can be reduced, while not interferingwith the pathway through which it acts. In this assay, cells receive afirst treatment with an siRNA to reduce RNase H1 mRNA, followed by asecond treatment with a known or putative RNase Hl-dependent compound.The target RNA cleavage following the second treatment is used to assesswhether the siRNA affected the enzyme activity stimulated by theaddition of the oligomeric compound.

A549 cells were treated with 100 nM of an siRNA directed to RNase H1,comprised of the antisense strand with the sequenceCUCAUCCUCUGUGGCAAACUU (SEQ ID NO: 218) annealed to the complementarysense strand (AAGUUUGCCACAGAGGAUGAG, SEQ ID NO: 219). Both strands areoligoribonucleotides with phosphodiester linkages throughout thecompounds. As controls, cells were treated with 100 nM of thesingle-strand sense RNA (SEQ ID NO: 219) or were left untreated.Following 10 hours of treatment, RNase H1 mRNA expression was measuredby quantitative real-time PCR and was reduced by 49% in cells treatedwith the RNase H1 siRNA. Untreated cells and cells treated with thecontrol siRNA showed no reduction in RNase H1 mRNA expression. Cellswere split into 96-well format cell culture plates at a density of 6000cells per well and were cultured for an additional 10 hours. Next, cellswere treated with ISIS 336848 (SEQ ID NO: 105) at 5, 10 or 30 nM. ISIS336848 is a gap-disabled compound targeted to C-raf and having the motif3-2-1-2-1-2-1-2-1-2-3; internucleoside linkages are phosphorothioatethroughout the compound and all cytosines are 5-methylcytosines. C-rafmRNA was measured by quantitative real-time PCR as described herein.Untreated cells served as the control to which data were normalized. Thedata are presented in Table 35 as percentage reduction in C-raf mRNA.

TABLE 35 Gap-disabled mediated reduction of C-raf mRNA in A549 cellswith lowered RNase H1 activity Reduction in C-raf mRNA Concentration ofgap-disabled compound (nM) 5 10 30 Single-strand sense RNA 40 61 89 NosiRNA 39 55 89 RNase H1 siRNA 32 48 83

These data demonstrate that, in comparison to cells treated with acontrol sense RNA or cells left untreated, the reduction in expressionof RNase H1 mRNA results in a decrease in the ability of thegap-disabled oligomeric compound to result in cleavage of its targetmRNA. For example, whereas a dose of 5 nM of ISIS 336848 results in 39%and 40% reductions in target mRNA in cells receiving no siRNA orsingle-strand sense RNA, respectively, C-raf mRNA is reduced by only 32%in cells in which RNase H1 has been reduced by siRNA treatment.

This assay was also performed in HeLa cells, in which either RNase H1 orRNase H2 was disrupted using siRNAs directed to the mRNA sequenceencoding each respective enzyme. The siRNA directed to RNase H1 wascomprised of SEQ ID NOs: 218 and 219. The siRNA directed to RNase H2 wascomprised of the antisense strand with the sequenceGGAGCCUUGCGUCCUGGGCTT (SEQ ID NO: 220), annealed to the complementarysense strand GCCCAGGACGCAAGGCTCCTT (SEQ ID NO: 221). SEQ ID NOs 220 and221 are oligoribonucleotides 19 nucleobases in length each having atwo-nucleobase overhang of deoxythymidine. In cells receiving no firsttreatment with siRNA, the second treatment with 5, 10 or 30 nM of ISIS336848 (gap-disabled) resulted in 18, 32 and 75% reductions in C-rafmRNA. In cells in which RNase H2 was disrupted, a second treatment with5, 10 or 30 nM of ISIS 336848 (gap-disabled) resulted in 23, 38 and 73%reductions in C-raf mRNA. Thus, disruption of RNase H2 did notsignificantly affect gap-disabled oligomeric compound activity. However,in cells in which RNase H1 was disrupted, a second treatment with 5, 10or 30 nM of the gap-disabled oligomeric compound resulted in 14, 28 and58% reductions in C-raf mRNA. These data illustrate that siRNA-mediatedreduction of RNase H1 mRNA reduced the potency of the gap-disabledcompound. Thus, gap-disabled compounds elicit target RNA cleavagethrough the activity of RNase H1.

Example 58 Overexpression of RNase H

In a further embodiment, RNase H overexpression in cultured cells wastested for its effects on the potency of gap-disabled oligomericcompounds. RNase H overexpression was accomplished using the RNase Hfull-length coding regions packaged in adenoviral vectors, which wereprepared as previously described (Wu et al., J. Biol. Chem., 2004, 279,17181-17189). An RNase H1 adenoviral vector was prepared using thefull-length RNase H1 coding region. An RNase H2 adenoviral vector wasprepared in the same manner, using the RNase H2 full-length codingregion. An additional vector was prepared using a truncated human RNaseH1 cDNA that encodes a protein lacking the 26 N-terminal amino acids;this construct is named RNase H1(−26). Two native isoforms of humanRNase H1 exist in the cell: a full length RNase H1 and the truncatedRNase Hl. The N-terminal 26 amino acids of human RNase H1 comprise amitochondial localization signal, thus the full-length isoform is foundpredominantly in the cytosol and mitochondria and the truncated protein(lacking the localization signal) is found predominantly in the nucleus.When compared in the in vitro assays described herein, both isoformsbehave similarly with respects to enzyme kinetics. A control vector,pLox, contained the shuttle vector used in preparation of the RNaseH-containing viruses and lacked the inserted genes.

In this assay, HeLa cells were cultured in DMEM supplemented with 10%fetal bovine serum, 0.005 mg/mL insulin, 0.005 mg/mL transferring, 5ng/mL selenium, 40 ng/mL dexamethasone (medium and all supplements fromInvitrogen Corporation, Carlsbad, Calif.). Cells were plated at adensity of approximately 6000 cells per well in 96-well plates andinfected with RNase H1, RNase H2, RNase H1(−26) or pLox adenovirus at200 plaque forming units per cell (pfu/cell). After 12 hours, cellstransfected with each virus were collected for RNA isolation andreal-time PCR quantitation of RNase H mRNA. The remaining cells weretransfected with the gap-disabled compound ISIS 336848 (SEQ ID NO: 105)at concentrations of 15, 30 and 45 nM, using LIPOFECTINT™ as describedherein. Cells were harvested 24 hours later, RNA was isolated and C-rafmRNA levels were measured using real-time PCR, as described herein.Real-time PCR measurements of RNase H1, RNase H2, RNase H1(−26) andC-raf were normalized using the housekeeping gene cyclophilin. Theresults of this assay are shown in Table 36. RNases H mRNA levels areshown as percent relative to RNase H expression in pLox-infected cells.C-raf mRNA levels are presented as percent reduction relative to cellsthat did not receive oligomeric compound treatment.

TABLE 36 Gap-disabled compound potency in cells overexpressing RNases HVirus pLox RNase H1 RNase H1(−26) RNase H2 RNase H 100% 2938% 801% 1216%level Dose of ISIS 336848 % Reduction in C-raf mRNA 15 nM 30 45 37 26 30nM 54 55 56 46 45 nM 63 67 74 60

These data demonstrate that when RNase H1 is present at a levelapproximately 30 times higher than that in pLox-infected cells,treatment with a 15 nM dose of the gap-disabled compound resulted in a45% reduction in C-raf mRNA, whereas target mRNA was reduced by only 30%in pLox-infected cells. Overexpression of RNase H1(−26) to levelsapproximately 8 times higher than that in p-Lox-infected cells alsoimproved the potency of the gap-disabled compound. An excess of RNase H2did not improve the activity of the gap-disabled compound. When the %reduction in C-raf mRNA is plotted against the base-10 logarithm of thegap-disabled compound concentration, an increase in gap-disabledcompound activity is apparent at all oligomeric compound concentrationstested in this assay. Similar observations were made in cells expressingany of the RNases H at levels 5 to 7 times that of the p-Lox-infectedcells. These data suggest that overexpression of either isoform improvesgap-disabled oligomeric compound activity and that gap-disabledoligomeric compounds are active in both the nucleus and cytosol. Thesedata further illustrate that gap-disabled compounds elicit target RNAcleavage through RNase Hi.

Example 59 In Vivo Analysis of Gap-Disabled Compounds Targeted to MouseGlucagon Receptor

In a further embodiment, gap-disabled chimeric oligomeric compoundstargeted to mouse glucagon receptor were tested for their effects ontarget reduction in vivo. The gap-disabled compounds were: ISIS 332866,ISIS 332868, ISIS 352426 and ISIS 352427 (all with the nucleotidesequence of SEQ ID NO: 43). Also tested were ISIS 180475 (SEQ ID NO:43), a gapmer compound; ISIS 332867, also a gapmer compound; ISIS 335032(SEQ ID NO: 43), an oligomeric compound uniformly comprised of2′-deoxynucleotides and ISIS 298683 (SEQ ID NO: 43), an oligomericcompound uniformly comprised of 2′-MOE nucleotides. The motif of eachcompound is shown in Table 37 as described for other compounds herein.Male Balb/c mice, 6 to 7 weeks of age, received twice weeklyintraperitoneal injections of approximately 1, 3 or 10 mg/kg of thecompounds shown in Table 37. Saline-injected animals served as a controlgroup and were injected in the same manner as the oligomeric compounds.Each treatment group contained 4 animals.

Liver RNA was analyzed for glucagon receptor expression levels byquantitative real-time PCR as described by other examples herein, usingthe housekeeping gene cyclophilin A to normalize RNA levels amongsamples. In Table 37, glucagon receptor mRNA expression levels are shownas percentage of saline-treated control glucagon receptor levels. Avalue less than or greater than 100 indicates a decrease or increase inmRNA expression, respectively. If present, “ND” indicates “notdetermined”.

TABLE 37 Target reduction following treatment with chimeric oligomericcompounds targeted to mouse glucagon receptor % Control SEQ Dose ofoligonucleotide ISIS # ID NO Motif 10 mg/kg 3 mg/kg 1 mg/kg 335032 43Uniform 2′-deoxy 10 85 117 332866 43 3-5-4-5-3 27 53 115 332867 433-14-3 12 111 112 332868 43 3-3-2-4-2-3-3 40 77 107 352426 43 2-6-4-6-221 66 115 352427 43 2-7-2-7-2 9 48 122 298683 43 Uniform 2′-MOE 84 ND ND180475 43 5-10-5 15 53 93

These results demonstrate that treatment with 3 mg/kg and 10 mg/kg dosesof the gap-disabled compounds ISIS 332866, ISIS 332868, ISIS 352426 andISIS 352427, in addition to the gapmer compound ISIS 180475 and theuniform 2′-deoxy compound ISIS 335032, inhibited mouse glucagon receptormRNA expression in a dose-dependent manner in vivo. ISIS 332867inhibited mouse glucagon receptor mRNA expression at the 10 mg/kg dose.

Example 60 In Vivo Analysis of Chimeric Oligomeric Compounds Targeted toFAS: Levin Rat Model

The Levin model is a polygenic model of rats selectively bred to developdiet-induced obesity (DIO) associated with impaired glucose tolerance,dyslipidemia and insulin resistance when fed a high-fat diet. Theadvantage of this model is that it displays traits more similar to humanobesity and glucose intolerance than in animals that areobese/hyperinsulinemic due to genetic defects, for example, a defect inleptin signaling. In a further embodiment, the gap-disabled compoundISIS 304170 (SEQ ID NO: 60), targeted to rat fatty acid synthase (FAS),was tested for its effects on target reduction in the Levin rat model.Male Levin rats were purchases from Charles River Laboratories atapproximately 8 weeks of age. Rats were fed a high-fat diet (60% fat)for 8 weeks, after which the animals were divided into three groups andtreated with saline, ISIS 304170 or the gapmer ISIS 256899 (SEQ ID NO:60). ISIS 256899, also targeted to rat FAS, was used as a positivecontrol for target reduction. Treatments were administeredsubcutaneously at a dose of 25 mg/kg, twice weekly, for 8 weeks. Controlgroups consisted of animals on the high-fat diet receiving salinetreatment and animals on a standard rodent diet receiving salinetreatment. Each treatment group included 5 to 6 animals.

At the end of the 8 week treatment period, animals were sacrificed andliver was collected for FAS protein analysis and white adipose tissue(WAT) and brown adipose tissue (BAT) were collected for measurement ofFAS mRNA expression. mRNA was measured by real-time PCR as describedherein and were normalized to levels in saline treated animals thatreceived a high-fat diet. Protein levels were measured by western blotas described herein, using an antibody recognizing rodent FAS (BDTransduction Laboratories of the BD Pharmingen Unit, San Diego, Calif.)and were normalized to levels in saline treated animals that received ahigh-fat diet. Data are shown in Table 38 and are expressed aspercentage of high-fat diet saline control. If present, “ND” indicates“not determined”. Also shown in Table 38 are serum cholesterol (mg/dL),triglyceride (mg/dL) and liver transaminase (ALT and AST, IU/L) levels,which were measured at the end of the study using routine clinicalanalyzer instruments (e.g. Olympus Clinical Analyzer, Melville, N.Y.).

TABLE 38 FAS, cholesterol and triglycerides in Levin rats treated withchimeric oligomeric compounds High High High Standard Fat Fat FatTreatment Saline Saline ISIS ISIS 304170 256899 % FAS protein, Liver 110100 55 39 % FAS mRNA, WAT ND 100 21 17 % FAS mRNA, BAT ND 100 5 6 SerumCholesterol (mg/dL) 81 81 69 160 Serum Triglyceride (mg/dL) 269 377 104607 ALT (IU/L) 79 73 158 61 AST (IU/L) 45 30 51 32

These data demonstrate that the gap-disabled compound ISIS 304170resulted in a marked reduction in rat FAS mRNA expression in both whiteand brown adipose tissue. Furthermore, rat FAS protein levels werereduced in liver following treatment with ISIS 304170. FAS mRNA andprotein levels were similar to those observed following treatment withthe gapmer compound. Furthermore, cholesterol and triglycerides in Levinrats receiving a high-fat diet were markedly lowered by treatment withISIS 304170, whereas ISIS 256899 did not lower cholesterol andtriglycerides in Levin rats receiving a high-fat diet. AST and ALTlevels were not at levels considered indicative of toxicity.

Plasma glucose concentrations were measured at 0 (beginning of study)and 8 (end of study) weeks of treatment by routine clinical analysisusing a Y512700 Select™ Biochemistry Analyzer (YSI Inc., Yellow Spring,Ohio). Plasma insulin levels were measured at 0 and 8 weeks of treatmentusing an insulin ELISA kit (ALPCO Diagnostics, Windham, N.H.) accordingto the manufacturer's instructions. Plasma leptin levels were measuredat 0 and 8 weeks using a rat leptin ELISA kit (Crystal Chem. Inc.,Downer's Grove, Ill.). Leptin is a hormone that regulates appetite.Plasma insulin, glucose and leptin levels are shown in Table 39.

TABLE 39 Plasma leptin, glucose and insulin in Levin rats treated withchimeric oligomeric compounds Diet and Treatment Study High High Highweek Standard Fat Fat Fat Saline Saline 304170 256899 Plasma Leptin(ng/mL) 0 11 42 46 46 Plasma Leptin (ng/mL) 8 10 57 8 24 Plasma Glucose(mg/dL) 0 106 103 100 105 Plasma Glucose (mg/dL) 8 95 115 106 98 PlasmaInsulin (ng/mL) 0 1.3 3.3 3.6 2.9 Plasma Insulin (ng/mL) 8 1.0 1.8 0.90.8

These data demonstrate that after 8 weeks, relative to animals receivinga high-fat diet and saline treatment, treatment with ISIS 304170 andISIS 256899 lowered plasma leptin, glucose and insulin levels. ISIS304170 lowered plasma leptin and insulin levels to those observed inrats on a standard diet. ISIS 256899 lowered plasma glucose levels tothose observed in rats on a standard diet.

After 4 weeks of treatment, an insulin tolerance test was performed.After 7 weeks of treatment, a glucose tolerance test was performed. Forthe tolerance tests, a baseline tail blood glucose measurement wasobtained, after which 1.0 g/kg glucose or 0.5 units/kg insulin wasadministered orally or intraperitoneally, respectively. Tail bloodglucose levels were measured using a Glucometer® instrument (AbbottLaboratories, Bedford, Mass.) at 15, 30, 60, 90, 120, 150 and 180minutes following the challenge with insulin and 30, 60, 90 and 120minutes following the glucose challenge. Insulin sensitivity in theanimals receiving ISIS 304170 and ISIS 256899 was similar to animals ona standard diet receiving saline treatment and was improved relative tosaline-treated animals on a high-fat diet. Glucose tolerance was notimproved by treatment with ISIS 304170 or ISIS 256899.

Body weight and food intake were measured weekly throughout the study.At the beginning of the study, animals on the high-fat diet weighedapproximately 670 grams. Whereas the saline-treated rats maintained thisweight throughout the treatment period, the body weights of rats treatedwith ISIS 304170 and ISIS 256899 dropped to approximately 500 g, thesame body weight as the rats on a standard rodent diet, by the end ofthe study. Throughout the study, food intake among rats treated withISIS 304170 and ISIS 256899 was approximately half that among salinetreated rats. Thus, concomitant with a reduction in FAS mRNA andprotein, body weight and food intake were lowered.

At the end of the study, liver and spleen weights, increases in whichcan indicate toxicity, were measured. Relative to saline-treated rats ona high-fat diet, liver weights were lower in rats receiving ISIS 304170and slightly higher in rats receiving ISIS 256899. The converse was truefor spleen weights, which were slightly raised in rats receiving ISIS304170. Fat depot weights were also determined. Treatment with ISIS304170 and ISIS 256899 prevented increases in brown adipose tissue andintra-abdominal white adipose tissue (both epididymal and perinephricfat) weights, which were all significantly raised in saline-treated ratson a high-fat diet.

Metabolic rate was measured after 4 weeks and 8 weeks of oligomericcompound treatment using indirect calorimetry in a metabolic chamber(Oxymax System, Columbus Instruments, Columbus, Ohio). No significantdifferenced in metabolic rates were observed when oligomericcompound-treated mice were compared to saline-treated mice.

This study in a rat model of diabetes and obesity illustrates thattreatment with the gap-disabled compound ISIS 304170 reduces rat FASprotein expression. Concomitant reductions are observed in body weight,fat depot weight, food intake, plasma leptin, plasma insulin, serumcholesterol, serum triglycerides and insulin sensitivity. Thus, thiscompound has applications in the treatment of diabetes, obesity andrelated conditions.

Example 61 In Vivo Analysis of Chimeric Oligomeric Compounds Targeted toFAS: Mouse Model of Diabetes and Obesity

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone lead to obesity in animals. ob/ob mice havea mutation in the leptin gene which results in obesity andhyperglycemia. As such, these mice are a useful model for theinvestigation of obesity and diabetes and treatments designed to treatthese conditions. ob/ob mice have higher circulating levels of insulinand are less hyperglycemic than db/db mice, which harbor a mutation inthe leptin receptor. In accordance with the present invention, theoligomeric compounds of the invention are tested in the ob/ob model ofobesity and diabetes.

Seven-week old male C57B1/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) were fed a diet with a fat content of approximately 11% andwere subcutaneously injected with ISIS 304171 (SEQ ID NO: 59) or ISIS148548 (SEQ ID NO: 59) at a dose of 25 mg/kg two times per week for 8weeks. Saline-injected animals served as a control group. Each treatmentgroup contained 8 mice.

After the treatment period, mice were sacrificed and FAS protein levelswere measured by western blot in liver and white adipose tissue (WAT),using an antibody that recognizes mouse FAS (BD TransductionLaboratories of the BD Pharmingen Unit, San Diego, Calif.). Relative toFAS protein levels in saline-treated mice, treatment with ISIS 304171reduced protein expression by 66% in liver and by 62% in white adiposetissue. Treatment with ISIS 148548 resulted in 93% and 81% reductions infatty acid protein in liver and white adipose tissue, respectively.

To assess the physiological effects resulting from reduction of FASexpression, the mice were further evaluated at the end of the treatmentperiod for serum triglycerides, serum cholesterol, and serumtransaminase levels. Triglycerides, cholesterol and transaminases weremeasured by routine clinical analyzer instruments (e.g. Olympus ClinicalAnalyzer, Melville, N.Y.). Triglyceride levels were 173, 101 and 118mg/dL in mice receiving treatment with saline, ISIS 304171 or ISIS148548, respectively. Cholesterol levels were 312, 221 and 218 mg/dL inmice receiving treatment with saline, ISIS 304171 or ISIS 148548,respectively. These data demonstrate that treatment with either thegap-disabled compound or gapmer compound targeted to FAS reduced bothcholesterol and triglyceride levels in ob/ob mice on a high fat diet.Also reduced were the liver transaminases ALT and AST (measured ininternational units/liter, or IU/L), indicating an improvement in liverfunction following treatment of ob/ob animals with a gap-disabled orgapmer compound. AST levels were 511, 277 and 334 IU/L in mice receivingtreatment with saline, ISIS 304171 or ISIS 148548, respectively. ALTlevels were 751, 481 and 344 IU/L in mice receiving treatment withsaline, ISIS 304171 or ISIS 148548, respectively.

Body weight was monitored throughout the study. Body weights in all 3treatment groups increased steadily throughout the first 5 weeks. After6, 7 and 8 weeks of treatment, the average body weight in ISIS304171-treated mice was 59 grams. The average body weight in ISIS148548-treated mice after 6, 7 and 8 weeks of treatment was 58 grams.However, in the saline-treated mice, the average body weight at 6, 7 and8 weeks was 64 grams. Thus, treatment with the gap-disabled or gapmercompound targeted to FAS resulted in reduced weight gain in ob/ob miceon a high fat diet.

Metabolic rate was measured after 5 weeks of oligomeric compoundtreatment using indirect calorimetry in a metabolic chamber (OxymaxSystem, Columbus Instruments, Columbus, Ohio). No significantdifferenced in metabolic rates were observed when oligomericcompound-treated mice were compared to saline-treated mice.

Adipose tissue weight was also measured at the end of the study. Nosignificant differences were observed when the oligomericcompound-treated mice were compared to saline-treated mice.

The effects of target inhibition on glucose metabolism were alsoevaluated. After 7 weeks of treatment with oligomeric compounds, an oralglucose tolerance test was performed. Mice received an oral dose ofapproximately 1 g/kg of glucose, and blood glucose levels were measuredat 30 minute intervals for up to 2 hours. Glucose levels are measuredusing a YSI glucose analyzer (YSI Scientific, Yellow Springs, Ohio). Nodifferences in glucose tolerance were observed when oligomericcompound-treated mice were compared to saline-treated mice.

These data demonstrate that the gap-disabled compound ISIS 304171, likethe gapmer compound ISIS 148548, reduced FAS expression in the livers ofob/ob mice. Furthermore, reductions were observed in serum cholesteroland triglycerides, body weight and liver transaminases.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference (including, but not limitedto, journal articles, U.S. and non-U.S. patents, patent applicationpublications, international patent application publications, gene bankaccession numbers, and the like) cited in the present application isincorporated herein by reference in its entirety.

1-67. (canceled)
 68. A chimeric oligomeric compound comprising fromabout 5 to about 80 linked nucleosides, wherein the chimeric oligomericcompound is divided into at least 5 separate regions, wherein each ofthe regions is a continuous sequence from 1 to about 5 nucleosides eachcomprising a 3′-endo sugar conformational geometry or a continuoussequence of from 1 to about 5 2′-deoxyribonucleosides, and wherein eachof the regions comprising from 1 to about 5 2′-deoxyribonucleosides isinternally located between two of the regions comprising 1 to about 5nucleosides each comprising a 3′-endo sugar conformational geometry orat one of the 3′ or 5′-termini.
 69. The compound of claim 68 comprising5 separate regions.
 70. The compound of claim 68 comprising 7 separateregions.
 71. The compound of claim 68 comprising 9 separate regions. 72.The compound of claim 68 comprising 11 separate regions.
 73. Thecompound of claim 68 comprising 13 separate regions.
 74. The compound ofclaim 68 comprising 15 separate regions.
 75. The compound of claim 68comprising 17 separate regions.
 76. The compound of claim 68 whereineach of the regions is from 1 to 4 nucleosides in length.
 77. Thecompound of claim 68 wherein each of the nucleosides comprising a3′-endo sugar conformational geometry is, independently, a sugarmodified nucleoside, a base modified nucleoside, or a nucleoside havingone or more modifications that include both the base and the sugar. 78.The compound of claim 77 wherein each of the nucleosides comprising a3′-endo sugar conformational geometry is a sugar modified nucleoside.79. The compound of claim 77 wherein each of the sugar modifiednucleosides, independently, comprises a 2′-substituent group.
 80. Thecompound of claim 79 wherein each of the 2′-substituent groups is,independently, O(CH₂)₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂ON(CH₃)₂,O(CH₂)₂—O—(CH₂)₂N(CH₃)₂, OCH₂C(═O)N(H)CH₃, OCH₃, O(CH₂)₂NH₂,O(CH₂)₂N(CH₃)₂, O(CH₂)₃NH₂, O(CH₂)₃N(H)CH₃, CH₂CH═CH₂, orO(CH₂)₂S(O)CH₃.
 81. The compound of claim 77 wherein at least one of thenucleosides of one of the regions comprising 3′-endo sugarconformational geometry has a bicyclic sugar moiety.
 82. The compound ofclaim 81 wherein at least one of the bicyclic sugar moieties is a lockednucleic acid (LNA).
 83. The compound of claim 68 comprising from about12 to 30 nucleosides in length.
 84. The compound of claim 68 comprisingfrom about 15 to 25 nucleosides in length.
 85. The compound of claim 68comprising from about 21 to 25 nucleosides in length.
 86. The compoundof claim 68 wherein each of the nucleosides is, independently, linked byphosphodiester or phosphorothioate.
 87. The compound of claim 68 whereineach of the nucleosides is linked by a phosphorothioate internucleosidelinking group.